Calcium influx-mediated signaling is required for complete mouse egg activation

Reproductive Medicine Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2012; 109(11):4169-74. DOI: 10.1073/pnas.1112333109
Source: PubMed


Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

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    • "We also have to take into consideration that the modulation of Ca 2+ spikes in mammalian oocytes is likely to be more complex, as the maintenance of Ca 2+ oscillations requires external Ca 2+ to replenish the intracellular Ca 2+ . In ICSI-induced mouse oocyte activation, this Ca 2+ influx across the plasma membrane, which is activated by the emptying of intracellular Ca 2+ stores following the first Ca 2+ transients, replenishes the Ca 2+ stores and also activates a cortical signalling pathway that is required for the actin-based functions necessary for meiotic spindle rotation and polar body extrusion (Miao et al., 2012). Mitochondria also participate in intracellular Ca 2+ homeostasis. "
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    ABSTRACT: In a recent report in Reproductive Biomedicine Online by Ebner et al., a comprehensive multi-centre study was presented on the use of a calcium ionophore, A23187, to artificially activate oocytes from patients who had poor fertilization rates in previous cycles. Under physiological conditions, the calcium increase in oocytes at activation is caused by influx and release from specific stores and ion channels, and has precise temporal, quantitative and spatial patterns. Calcium ionophores may release Ca(2+) in an uncontrolled fashion from intracellular stores that would not normally be involved in the activation process. Ionophores, including A23187, have a multitude of effects on cell homeostasis, not yet defined in oocytes, that may have long-term effects, for example on gene expression. We suspect that the successful births reported by Ebner et al. are a result of the overriding influence of the injected spermatozoa, rather than the effect of the ionophore; nevertheless, such an invasive non-physiological approach to assisted reproduction techniques is worrying, especially as epigenetic effects may result in future generations. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
    Full-text · Article · Jul 2015 · Reproductive biomedicine online
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    • "However, Ca 2+ overload alone, as we observed with TRPV3 stimulation, also terminates Ca 2+ oscillations. Recent studies suggest that CRAC channels are not functional in mouse eggs (Miao et al., 2012; Takahashi et al., 2013). We evaluated the pattern of PLCz-induced calcium oscillations in V3-KO and Het-V3 eggs in the presence of the CRAC blocker STA-12-5775 (RO2959; 3 mM; Chen et al., 2013). "
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    ABSTRACT: In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3(-/-) eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3(-/-) eggs failed to conduct Sr(2+) or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.
    Full-text · Article · Dec 2013 · Cell Reports
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    • "It should also be noted that ethanol activation is induced in a complete medium whereas Ca2+ ionophore activation is induced in a Ca2+-free medium. In this regard, it has been recently shown that external Ca2+ entry is needed for complete egg activation [30], supporting the idea that the source of Ca2+ is important for PS exposure. Because inseminated ionophore-activated eggs are able to expose PS other factors may also be involved in PS exposure. "
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    ABSTRACT: Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca(2+) concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca(2+) spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca(2+) release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca(2+) ionophore, suggesting that the Ca(2+) source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca(2+) rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.
    Full-text · Article · Aug 2013 · PLoS ONE
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