Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

Abstract

Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.

Figure 1. Vital zinc probe reveals cortical compartments in the female gamete in mouse

(a) Final step (see methods) in the synthesis of ZincBY-1, a novel fluorescent zinc probe. (b) Fluorescence emission of ZincBY-1 in EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid)-buffered Zn²⁺ solutions. Error bars represent ± S.E.M. (standard error of the mean). Spectra acquired in 100 mM KNO₃, 50 mM HEPES, pH 7.2, λ_ex = 520 nm. Integrated emission (530–700 nm) plotted vs. calculated [Zn²⁺_free] and fitted to apparent K_d = 2.5 nM. (c) GV (germinal vesicle) oocyte, MII (metaphase II) egg, and parthenote incubated with 50 nM ZincBY-1 (green) and DNA probe Hoechst 33342 (blue). Representative Z-stack projection (i, iv, vii), confocal optical slice (ii, v, viii), and brightfield (iii, vi, ix) images shown. Bright, punctate, cortical fluorescence from ZincBY-1 is observed in GV and MII cells. (d) Incubation with 10 µM TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) for 10 minutes abolishes cortical fluorescence in ZincBY-1 stained cells. (e) Distance of ZincBY-1 compartments from the plasma membrane in GV (black) and MII (grey) cells. Vesicle positions sorted into 1 µm bins and plotted as a histogram. >90% of compartments are within 5 µm of the membrane in both cell types. All scale bars = 20 µm

Figure 2. Labile zinc is cortically localized in the oocyte and tracks with cortical granule staining

(a) Schematic of meiotic maturation. GV oocytes in the ovary are arrested at prophase I (PI) of meiosis. Upon hormonal signaling, maturation begins and the cell progresses through meiosis until it arrests at metaphase II (MII), at which point the egg is competent for fertilization. Intervening stages during maturation include germinal vesicle breakdown (GVBD), metaphase I (MI) and Telophase I/Anaphase I (TI/AI). (b) (i–v) Samples at GV, GVBD, MI, AI, MII were labeled with the zinc-specific probe ZincBY-1 (50 nM, green) to track labile zinc and counterstained with Hoechst 33342 to label DNA (blue). (vi–x) Following zinc imaging, cells were fixed and stained for cortical granules using fluorescently labeled Lens culinaris agglutinin (LCA, red) and counterstained for DNA with DAPI (blue). Staining patterns for zinc and LCA are similar at each stage of maturation, suggesting that zinc-enriched structures may represent the same vesicles as, or comprise a subpopulation of the cortical granules. Representative optical confocal sections for each meiotic stage is shown. At least five oocytes were visualized at each stage in three independent experiments. Scale bars = 25 µm. * represents an adjacent oocyte.

Figure 3. Zinc fixation enables ultrastructural identification of zinc-enriched cortical compartments by Scanning Transmission Electron Microscopy with Energy Dispersive Spectroscopy (STEM-EDS)

(a) Zinc-fixation schematic. Eggs were fixed and treated with NaHS to form ZnS. Following ethanol dehydration and resin embedding, eggs were used intact for X-ray fluorescence (XFM) tomography or sectioned prior to STEM-EDS or XFM Bionanoprobe analysis. (b) Diagram of STEM microscope with dual EDS detectors for zinc mapping. (c) Z-contrast image of a 200 nm section of a resin-embedded MII egg following zinc fixation. Vesicles, zona pellucida (ZP), plasma membrane (PM) and ooplasm indicated. Bright and dark areas indicate regions with high and low molecular weight content respectively. Bright signal is concentrated in cortical compartments. Scale bar = 0.5 µm. (d) Histogram of diameters of cortical compartments in STEM-EDS samples (20 nm bins). Distribution centers on a diameter of ~260 nm. Data from 23 Zn-enriched compartments from 8 eggs. (e) EDS spectra of bright compartment (blue) and cytoplasm (red). Zinc signal is enriched in the compartment relative to the cytoplasm. (f) Z-contrast, Zn and S EDS maps of a cortical region in a MII egg. Overlay demonstrates that Zn-rich regions correspond to areas high in S and electron density (Z-contrast).

Figure 4. X-ray fluorescence microscopy and tomography provides zinc quantification and mapping within the egg

(a) Bionanoprobe XFM images of a 400 nm thick egg section. Zn, Cu, and Fe maps shown with concentration ranges. Scale bar = 10 µm, pixels = 100 × 100 nm². High [Zn] observed in punctate cortical structures. (b) Histogram of [Zn] in punctate regions (bins = 0.05 M). Data fitted to a Lorentzian distribution (black line) centered on [Zn] = 0.2 M. (c) XFM tomography images at 0° angle of intact MII egg following zinc fixation. Zn (i), S (ii), Cu (iv), Fe (v), and Ca (vi) maps shown with concentration ranges. Zn/S map overlay (iii) demonstrates zinc-rich regions are intracellular. Scale bar = 20 µm. (d) Total metal content quantification in resin-embedded MII egg following zinc fixation. Bars represent average number of atoms over 60 projection images of the same sample (Fe = 8±4 × 10⁹; Cu = 5±3 × 10⁹; Zn = 6±2 × 10¹⁰). Error bars represent ± S.E.M. Dashed lines represent previously measured values in unfixed eggs. Results indicate that this zinc fixation protocol preserves total Zn content. (e) Zn maps at several angles (° indicated) illustrate a cortical, hemispherical distribution of Zn-enriched regions.

Figure 5. Live cell fluorescence zinc imaging demonstrates intracellular zinc compartments are the source of the extracellular zinc spark

MII eggs labeled with 50 nM ZincBY-1 (intracellular, green) were activated with 10 mM SrCl₂ in medium containing 50 µM FluoZin-3 (extracellular, red). All scale bars = 20 µm. (a–c) Whole egg was imaged in a z-stack time course (5 µm optical sections taken over 6.5 seconds). (a) Z-stack projections of ZincBY-1 and FluoZin-3 fluorescence during a zinc spark. Arrows indicate concentrated regions of zinc exocytosis. (b) Overlaid optical sections of pre-spark ZincBY-1 fluorescence and FluoZin-3 fluorescence during a zinc spark. Z-stack position is indicated in each panel. (c) Angular analysis of intracellular and extracellular fluorescence distribution in a z-section (z14 shown, others in SI). Fluorescence intensity pattern is the same in both channels, indicating that zinc-enriched vesicles are the source of the zinc spark. (d–e) Egg imaged in a 1 µm confocal section. (d) Images from time course taken before (i), during (ii), and after (iii) a zinc spark. Brightfield image indicates intracellular and extracellular ROIs. (e) Time traces show simultaneous decrease in intracellular fluorescence and increase in extracellular fluorescence, indicating that zinc-enriched vesicles are the source of the zinc spark.

Figure 6. The zinc flux during egg activation is regulated by a quantitative loss of cortical zinc compartments

This schematic diagram summarizes the fluxes in total zinc that are observed during maturation and immediately following fertilization/egg activation. The meiotic cycle in the mammalian egg occurs over a very short period of time that is accompanied by a very large flux in the amount and localization of the inorganic element zinc (black bars).^, Through the combined used of our novel zinc probe ZincBY-1 for live cell imaging and a suite of fixed cell imaging technologies (sulfide-zinc fixation, STEM-EDS, Bionanoprobe XFM, XFM tomography), we have estimated that there are 8,000 zinc-enriched cortical vesicles in the egg, each containing ~1 million zinc atoms. These vesicles, containing in total 8 billion zinc atoms, are lost at fertilization during the zinc spark and quantitatively contribute to zinc efflux that is required during the egg-to-embryo transition. This study sets a precedent for how zinc can be quantitatively tracked during key biological processes.