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Graphical Abstract
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Figure 2:. Elemental analysis of eggs and embryos.ICP-MS measures a significant decrease in intracellular manganese post-fertilization. a. Metal concentrations of Xenopus eggs and embryos (1 hour post-fertilization) determined by ICP-MS. Intracellular Co and Ni are both below 13 μM. Data are presented as mean ± SEM per egg/embryo, **: p = 0.0081. Each point corresponds to ICP-MS analysis of a batch of 20 eggs normalized to a per egg basis. Similar results were obtained in 6 separate experiments (not shown). b. Elemental contents (mM) of Xenopus eggs and embryos (1-hour post-fertilization). Experimental conditions as in a. In both a. and b. n = samples from 4 separate frogs analyzed in a single ICP experiment. p is the significance of the change in elemental content following fertilization. Two-tailed, heteroscedastic T-tests were run between eggs and embryos in order to determine if there was a significant difference in metal content. No adjustments were made for multiple comparisons.
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Figure 3:. Paramagnetic resonance measurements of eggs and embryos.EPR confirms a post-fertilization decrease in manganese, while EPR, ENDOR, and ESEEM demonstrate that the majority of intracellular manganese is bound to a low molecular weight carboxylate. 35-GHz, 2K EPR/ENDOR/ESEEM Spectra: a. Representative absorption-display CW-EPR spectra of Mn2+ in: frog eggs (black); embryos (red; 1/3 amplitude of eggs, ~30 ± 10% remaining, see text); Mn-orthophosphates Mn-Pi (blue; scaled to eggs); EPR spectra of frog eggs/Mn-Pi offset by +1,350/+2,350. Inset: digital derivatives of the spectra, accentuating the six-line 55Mn hyperfine pattern around 12kG magnetic field. b.
31P/1H Davies pulsed-ENDOR spectra of: intact frog eggs and embryos; solution Mn2+ complexes (aquo [H2O], orthophosphate [Pi], polyphosphate [polyP]). Braces represent frequency ranges of 31P and 1H ENDOR signals. ENDOR spectra are normalized to Mn2+ concentration; thus 31P%, 1H% peaks (gray highlight) represent absolute ENDOR responses. Insets: (a) 31P% ENDOR responses of exemplars and eggs/embryos from frog 3; (b) corresponding 1H% ENDOR responses. (* is a 55Mn ENDOR signal) c.
14N 3-pulse ESEEM timewaves of: eggs (black); embryos (red), each multiplied by 5 for ease of comparison; solution Mn-imidazole complex (purple). Experimental conditions: see Supplemental Methods (b, c, Fields ~ 12.5k G). In a., b., and c. n = 3 biologically independent replicates of intact frog eggs and embryos examined over 3 independent experiments.
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Figure 4:. Synchrotron-based X-ray fluorescence mapping of egg and embryo cortices.X-ray fluorescence microscopy images of the animal pole of fixed Xenopus egg/embryo slices show zinc and manganese are localized in small cortical compartments. Scale bar = 20 μm. Images acquired at Beamline 2-ID-D at Argonne National Laboratory. Pixel size: 300 × 300 nm, slice thickness: 2 μm, scan time: 500 ms/pixel. These are representative images of slices of eggs/embryos from 7 different frogs imaged over 3 experiments.
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Figure 5:. Animal pole egg metal compartment contents.Metals are stored at millimolar concentrations in the cortical compartments. a. Concentrations of metal compartments in the egg animal pole. Voxel size 300 × 300 × 400 nm. Data acquired at Beamline 2-ID-D, Argonne National Laboratory. Values reported as mean ± SD, n = 3 slices of eggs analyzed from separate frogs examined over 2 independent experiments. b. Representative image of zinc distribution and thresholding of areas of high zinc concentration. Scale bar = 10 μm. Images acquired at Beamline 2-ID-D at Argonne National Laboratory. Pixel size: 300 × 300 nm, slice thickness: 400 nm, scan time: 500 ms/pixel. c. Representative plot of the zinc concentration distribution of small metal compartments found in a slice. n = 156 compartments.
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Figure 6:. Analytical electron microscopic analysis of egg animal pole cortical vesicles.AEM demonstrates that multiple metals are stored in sub-micrometer vesicles in the animal pole. a. High Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM) images of the cortex of the animal pole of a fixed Xenopus egg. b. Hyperspectral elemental intensity distribution maps: background corrected, with a 0.7-pixel gaussian blur, no normalization, no quantification. Same conditions as in a. Scale bar = 1 μm in both a. and b. These are representative images of 3 separate scans. c. Cumulative XEDS spectra confirming the presence of Ca, Mn, Co, Ni, Cu, and Zn in vesicles. These comparison spectra are obtained by summing individual spectra from an identical number of pixels in regions of interest which correspond to vesicle and neighboring non-vesicle areas. d. Histogram of vesicle diameter distribution from image in a. n = 50 vesicles measured.
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Figure 7:. Extracellular zinc or manganese inhibits fertilization in a dose-dependent manner.Both extracellular zinc and manganese block fertilization. a. Images of an unfertilized Xenopus egg (in jelly coat), a properly dividing embryo (first cleavage), and an instance of failed cleavage (meiotic catastrophe). b. An IC50 graph of the effects of extracellular ZnSO4 on the rate of fertilization. IC50 = 53 μM (95% CI = 47 – 61 μM, R2 = 0.94). Data are presented as mean percentage of eggs properly fertilized ± SEM, n = 4 frogs (39 – 102 eggs per datapoint). See Supplemental Figure 11 for raw data. c. An IC50 graph of the effects of extracellular MnCl2 on the rate of fertilization. IC50 = 890 μM (95% CI = 770 μM – 1.0 mM, R2 = 0.93). Data are presented as mean percentage of eggs properly fertilized ± SEM, n = 4 frogs (36 – 100 eggs per datapoint). See Supplemental Figure 12 for raw data.
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