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Metal ion fluxes controlling amphibian fertilization


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.

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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.

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,; XFM data for Figs. 4 and 5 and Supplementary Figs. 4–6,; AEM data for Fig. 6 and Supplementary Figs. 7–9,; TEM cortical images for Supplementary Fig. 10, Source data are provided with this paper.


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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




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 or Teresa K. Woodruff or Thomas V. O’Halloran.

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Competing interests

The authors declare no competing interests.

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

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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.

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Seeler, J.F., Sharma, A., Zaluzec, N.J. et al. Metal ion fluxes controlling amphibian fertilization. Nat. Chem. 13, 683–691 (2021).

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