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: 9.81). 02/2012; 109(11):4169-74. DOI: 10.1073/pnas.1112333109
Source: PubMed

ABSTRACT 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.
    Reproductive biomedicine online 07/2015; 21. DOI:10.1016/j.rbmo.2015.06.013 · 2.98 Impact Factor
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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.
    Cell Reports 12/2013; 5(5). DOI:10.1016/j.celrep.2013.11.007 · 8.36 Impact Factor
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    • "Subcellular localization of the Ca 2+ signal involved in PB2 emission appears to be critical for its success. Blocking Ca 2+ influx in fertilized mouse eggs prevents PB2 emission, despite the occurrence of multiple intracellular Ca 2+ oscillations and cell cycle resumption; the end result of this treatment is the formation of embryos containing three pronuclei (Miao et al. 2012). "
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    ABSTRACT: Calcium (Ca(2+) ) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca(2+) signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca(2+) sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca(2+) in many cell types and of the impact of cellular localization on Ca(2+) signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca(2+) is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca(2+) release and effectors of Ca(2+) signals. We then summarize studies exploring how Ca(2+) directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca(2+) signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe for future research. Mol. Reprod. Dev. 2012. This article is a US government work and, as such, is in the public domain in the United States of America.
    Molecular Reproduction and Development 11/2012; 79(11). DOI:10.1002/mrd.22078 · 2.68 Impact Factor
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