Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Metal ion fluxes controlling amphibian fertilization

Nature Chemistry volume 13, pages 683–691 (2021)Cite this article

Subjects

Abstract

Mammalian oocytes undergo major changes in zinc content and localization to be fertilized, the most striking being the rapid exocytosis of over 10 billion zinc ions in what are known as zinc sparks. Here, we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese. Synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through electron–nuclear double-resonance studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs, but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Zinc release from frog oocytes.
Fig. 2: Elemental analysis of eggs and embryos.
Fig. 3: Paramagnetic resonance measurements of eggs and embryos.
Fig. 4: Synchrotron-based XFM of egg and embryo cortices.
Fig. 5: AP egg metal compartment contents.
Fig. 6: AEM analysis of egg AP cortical vesicles.
Fig. 7: Extracellular zinc or manganese inhibits fertilization in a dose-dependent manner.

Similar content being viewed by others

Data availability

Data supporting the findings of this study are available within the Article and its Supplementary Information. The datasets generated and analysed during the current study are publicly available in the figshare repository, at the following links: confocal data for Fig. 1, Supplementary Fig. 1 and Supplementary Videos 1 and 2, https://figshare.com/articles/dataset/Confocal_Images/14233235; XFM data for Figs. 4 and 5 and Supplementary Figs. 4–6, https://figshare.com/articles/dataset/XFM_Data/14265332; AEM data for Fig. 6 and Supplementary Figs. 7–9, https://figshare.com/articles/dataset/AEM_Data/14265350; TEM cortical images for Supplementary Fig. 10, https://figshare.com/articles/dataset/TEM_Cortical_Images/12827894/1. Source data are provided with this paper.

References

  1. Kim, A. M., Vogt, S., O’Halloran, T. V. & Woodruff, T. K. Zinc availability regulates exit from meiosis in maturing mammalian oocytes. Nat. Chem. Biol. 6, 674–681 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kim, A. M. et al. Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs. ACS Chem. Biol. 6, 716–723 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Que, E. L. et al. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks. Nat. Chem. 7, 130–139 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Que, E. L. et al. Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation. Theriogenology 127, 41–48 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Duncan, F. E. et al. The zinc spark is an inorganic signature of human egg activation. Sci. Rep. 6, 24737 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kong, B. Y. et al. Maternally-derived zinc transporters ZIP6 and ZIP10 drive the mammalian oocyte-to-egg transition. Mol. Hum. Reprod. 20, 1077–1089 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Que, E. L. et al. Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy. Integr. Biol. (Camb.) 9, 135–144 (2017).

    Article  CAS  Google Scholar 

  8. Kong, B. Y. et al. The inorganic anatomy of the mammalian preimplantation embryo and the requirement of zinc during the first mitotic divisions. Dev. Dyn. 244, 935–947 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang, N., Duncan, F. E., Que, E. L., O’Halloran, T. V. & Woodruff, T. K. The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development. Sci. Rep. 6, 22772 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mendoza, A. D., Woodruff, T. K., Wignall, S. M. & O’Halloran, T. V. Zinc availability during germline development impacts embryo viability in Caenorhabditis elegans. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 191, 194–202 (2017).

    Article  CAS  PubMed  Google Scholar 

  11. Hu, Q. et al. Zinc dynamics during Drosophila oocyte maturation and egg activation. iScience 23, 101275 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Falchuk, K. H., Montorzi, M. & Vallee, B. L. Zinc uptake and distribution in Xenopus laevis oocytes and embryos. Biochemistry 34, 16524–16531 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Nomizu, T., Falchuk, K. H. & Vallee, B. L. Zinc, iron and copper contents of Xenopus laevis oocytes and embryos. Mol. Reprod. Dev. 36, 419–423 (1993).

    Article  CAS  PubMed  Google Scholar 

  14. de Laat, S. W., Buwalda, R. J. & Habets, A. M. Intracellular ionic distribution, cell membrane permeability and membrane potential of the Xenopus egg during first cleavage. Exp. Cell. Res. 89, 1–14 (1974).

    Article  PubMed  Google Scholar 

  15. Snow, P., Yim, D. L., Leibow, J. D., Saini, S. & Nuccitelli, R. Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs. Dev. Biol. 180, 108–118 (1996).

    Article  CAS  PubMed  Google Scholar 

  16. Stith, B. J. Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development. Dev. Biol. 401, 188–205 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fontanilla, R. A. & Nuccitelli, R. Characterization of the sperm-induced calcium wave in Xenopus eggs using confocal microscopy. Biophys. J. 75, 2079–2087 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lindsay, L. L. & Hedrick, J. L. Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening. Biochem. Biophys. Res. Commun. 324, 648–654 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Marszalek, I. et al. Revised stability constant, spectroscopic properties and binding mode of Zn(ii) to FluoZin-3, the most common zinc probe in life sciences. J. Inorg. Biochem. 161, 107–114 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Fahrni, C. J. & O’Halloran, T. V. Aqueous coordination chemistry of quinoline-based fluorescence probes for the biological chemistry of zinc. J. Am. Chem. Soc. 121, 11448–11458 (1999).

    Article  CAS  Google Scholar 

  21. Smirnova, J. et al. Copper(i)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agent. Sci. Rep. 8, 1463 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Auld, D. S., Falchuk, K. H., Zhang, K., Montorzi, M. & Vallee, B. L. X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis. Proc. Natl Acad. Sci. USA 93, 3227–3231 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gunter, T. E. et al. Determination of the oxidation states of manganese in brain, liver and heart mitochondria. J. Neurochem. 88, 266–280 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Ducic, T. et al. X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons. J. Neurochem. 124, 250–261 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Tsednee, M. et al. Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions. J. Biol. Chem. 294, 17626–17641 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McNaughton, R. L. et al. Probing in vivo Mn2+ speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy. Proc. Natl Acad. Sci. USA 107, 15335–15339 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sharma, A. et al. Across the tree of life, radiation resistance is governed by antioxidant Mn2+, gauged by paramagnetic resonance. Proc. Natl Acad. Sci. USA 114, E9253–E9260 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sharma, A. et al. Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to gamma-radiation by advanced paramagnetic resonance methods. Proc. Natl Acad. Sci. USA 110, 5945–5950 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Horning, K. J., Caito, S. W., Tipps, K. G., Bowman, A. B. & Aschner, M. Manganese is essential for neuronal health. Annu. Rev. Nutr. 35, 71–108 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tan, X., Bernardo, M., Thomann, H. & Scholes, C. P. Pulsed and continuous wave electron nuclear double resonance patterns of aquo protons coordinated in frozen solution to high spin MN2+. J. Chem. Phys. 98, 5147–5157 (1993).

    Article  CAS  Google Scholar 

  31. Dworkin, M. B. & Dworkin-Rastl, E. Carbon metabolism in early amphibian embryos. Trends Biochem. Sci. 16, 229–234 (1991).

    Article  CAS  PubMed  Google Scholar 

  32. Chen, S. et al. Synchrotron-based X-ray fluorescence microscopy as a complementary tool to light microscopy/electron microscopy for multi-scale and multi-modality analysis. Microsc. Microanal. 24, 86–87 (2018).

    Article  CAS  Google Scholar 

  33. Timm, F. Zur Histochemie der Schwermetalle das Sulfid-Silberverfahren. Dtsch. Z. Gesamte Gerichtl. Med. 46, 706–711 (1958).

    CAS  PubMed  Google Scholar 

  34. Danscher, G., Stoltenberg, M., Bruhn, M., Søndergaard, C. & Jensen, D. Immersion autometallography: histochemical in situ capturing of zinc ions in catalytic zinc–sulfur nanocrystals. J. Histochem. Cytochem. 52, 1619–1625 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Grey, R. D., Wolf, D. P. & Hedrick, J. L. Formation and structure of the fertilization envelope in Xenopus laevis. Dev. Biol. 36, 44–61 (1974).

    Article  CAS  PubMed  Google Scholar 

  36. Montorzi, M., Falchuk, K. H. & Vallee, B. L. Vitellogenin and lipovitellin: zinc proteins of Xenopus laevis oocytes. Biochemistry 34, 10851–10858 (1995).

    Article  CAS  PubMed  Google Scholar 

  37. Imoh, H. Establishment and movement of egg regions revealed by the size class of yolk platelets in Xenopus laevis. Rouxs Arch. Dev. Biol. 205, 128–137 (1995).

    Article  PubMed  Google Scholar 

  38. Zaluzec, N. J. Thin film characterization using analytical electron microscopy. Thin Solid Films 72, 177–192 (1980).

    Article  CAS  Google Scholar 

  39. Kotani, M., Ikenishi, K. & Tanabe, K. Cortical granules remaining after fertilization in Xenopus laevis. Dev. Biol. 30, 228–232 (1973).

    Article  CAS  PubMed  Google Scholar 

  40. Campanella, C. & Andreuccetti, P. Ultrastructural observations on cortical endoplasmic reticulum and on residual cortical granules in the egg of Xenopus laevis. Dev. Biol. 56, 1–10 (1977).

    Article  CAS  PubMed  Google Scholar 

  41. Benau, D. A., McGuire, E. J. & Storey, B. T. Further characterization of the mouse sperm surface zona-binding site with galactosyltransferase activity. Mol. Reprod. Dev. 25, 393–399 (1990).

    Article  CAS  PubMed  Google Scholar 

  42. Wozniak, K. L. et al. Zinc protection of fertilized eggs is an ancient feature of sexual reproduction in animals. PLoS Biol. 18, e3000811 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Werst, M. M., Davoust, C. E. & Hoffman, B. M. Ligand spin densities in blue copper proteins by Q-band proton and nitrogen-14 ENDOR spectroscopy. J. Am. Chem. Soc. 113, 1533–1538 (1991).

    Article  CAS  Google Scholar 

  44. Davoust, C. E., Doan, P. E. & Hoffman, B. M. Q-band pulsed electron spin-echo spectrometer and its application to ENDOR and ESEEM. J. Magn. Reson. A 119, 38–44 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research is supported by NIH grants R01GM115848 (T.V.O. and T.K.W.), R01GM038784 and P41GM181350 (T.V.O.) and R01GM111097 (B.M.H.). J.F.S. was supported by The Cellular and Molecular Basis of Disease Training Program at Northwestern University (NIH T32GM008061), N.J.Z. was supported by both LDRD funding no. 2017-153-N0 and the Photon Science Division at Argonne National Laboratory. X-ray fluorescence microscopy was performed at the Advanced Photon Source (APS), while analytical electron microscopy was performed using the ANL PicoProbe as well as AEM resources in the Center for Nanoscale Materials (CNM), both of which are Office of Science user facilities at Argonne National Laboratory. Use of the APS and CNM was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. This work made use of the BioCryo facility of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205), the MRSEC programme (NSF DMR-1720139) at the Materials Research Center, the International Institute for Nanotechnology (IIN) and the State of Illinois. Microscopy was performed at the Biological Imaging Facility at Northwestern University (RRID: SCR_017767), supported by the Chemistry for Life Processes Institute, the NU Office for Research and the Department of Molecular Biosciences. Elemental analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center supported by the Office of the Director, National Institutes of Health, via NIH grants S10OD026786 and S10OD020118. We thank R. Woodruff and L. Gross for the initial discovery of zinc fluxes in frog eggs, J. Hornick and S. Garwin for assistance with imaging, K. MacRenaris, O. Ali and R. Sponenburg for assistance with ICP-MS and P. Huber for assistance with Xenopus experiments.

Author information

Authors and Affiliations

  1. Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA

    John F. Seeler, Brian M. Hoffman, Carole LaBonne, Teresa K. Woodruff & Thomas V. O’Halloran

  2. Department of Chemistry, Northwestern University, Evanston, IL, USA

    Ajay Sharma, Emma G. Schultz, Brian M. Hoffman & Thomas V. O’Halloran

  3. Photon Sciences Directorate, Argonne National Laboratory, Argonne, IL, USA

    Nestor J. Zaluzec

  4. Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA

    Reiner Bleher

  5. Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA

    Barry Lai

  6. The Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Emma G. Schultz, Carole LaBonne, Teresa K. Woodruff & Thomas V. O’Halloran

  7. Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Teresa K. Woodruff

  8. Department of Obstetrics and Gynecology, Michigan State University, East Lansing, MI, USA

    Teresa K. Woodruff

  9. Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Thomas V. O’Halloran

  10. Department of Chemistry, Michigan State University, East Lansing, MI, USA

    Thomas V. O’Halloran

  11. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA

    Thomas V. O’Halloran

Authors
  1. John F. Seeler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ajay Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nestor J. Zaluzec
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Reiner Bleher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Barry Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Emma G. Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Brian M. Hoffman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carole LaBonne
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Teresa K. Woodruff
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Thomas V. O’Halloran
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.F.S., A.S., N.J.Z., B.M.H., C.L., T.K.W. and T.V.O. designed the research. J.F.S., A.S., N.J.Z., R.B. and E.G.S. performed the research. B.L. helped design and implement XFM experiments and process and analyse the data. J.F.S., A.S., B.M.H., T.K.W. and T.V.O. wrote the manuscript. All the authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Carole LaBonne, Teresa K. Woodruff or Thomas V. O’Halloran.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods, calculations, Figs. 1–12, Tables 1–4 and captions for Videos 1–6.

Reporting Summary

Supplementary Video 1

Zinc spark following fertilization of a Xenopus egg (time as mm:ss). The video was contrast-adjusted. Brightness and contrast were adjusted.

Supplementary Video 2

Zinc spark following parthenogenic activation of a Xenopus egg by ionomycin (time as mm:ss). The video was contrast-adjusted. Brightness and contrast were adjusted.

Supplementary Video 3

Control eggs in 0.1x MMR buffer with 1% DMSO (time as mm:ss).

Supplementary Video 4

Eggs treated with 20 μM ionomycin (time as mm:ss).

Supplementary Video 5

Eggs treated with 10 mM 1,10-phenanthroline (time as mm:ss).

Supplementary Video 6

Eggs treated with 10 mM ammonium tetrathiomolybdate (time as mm:ss).

Supplementary Data 1

Source data for Supplementary Figs. 1b and 1c.

Supplementary Data 2

Source data for Supplementary Fig. 2a.

Supplementary Data 3

Source data for Supplementary Fig. 3.

Supplementary Data 4

Source data for Supplementary Fig. 6a.

Supplementary Data 5

Source data for Supplementary Fig. 11.

Supplementary Data 6

Source data for Supplementary Fig. 12.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1c and 1d.

Source Data Fig. 2

Statistical source data for Fig. 2.

Source Data Fig. 3

Statistical source data for Fig. 3.

Source Data Fig. 5

Statistical source data for Fig. 5c.

Source Data Fig. 6

Statistical source data for Fig. 6d.

Source Data Fig. 7

Statistical source data for Fig. 7b and 7c.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Seeler, J.F., Sharma, A., Zaluzec, N.J. et al. Metal ion fluxes controlling amphibian fertilization. Nat. Chem. 13, 683–691 (2021). https://doi.org/10.1038/s41557-021-00705-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-021-00705-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing