Wozniak KL , Bainbridge RE , Summerville DW , Tembo M , Phelps WA , Sauer ML , Wisner BW , Czekalski ME , Pasumarthy S , Hanson ML , Linderman MB , Luu CH , Boehm ME , Sanders SM , Buckley KM , Bain DJ , Nicotra ML , Lee MT , Carlson AE .

R01 GM125638 NIGMS NIH HHS , R00 HD069410 NICHD NIH HHS , T32 HD087194 NICHD NIH HHS

             prevention of polyspermy
             positive regulation of fertilization

	Fig 1. X. laevis eggs release zinc at fertilization, which protects eggs from additional fertilizations. (A) Representative fluorescence and bright-field images of X. laevis eggs in FluoZin-3 before and after sperm application. Time is relative to sperm addition. Zinc release coincided with lifting of the envelope (red arrowheads and insert) (N = 16 eggs, 4 trials). (B) Integrated fluorescence relative to time of sperm addition and detected by region of interest analysis, indicated by colored boxes in panel (A). (C) Representative images of X. laevis eggs and embryos. (D) Box plot distribution of zinc ions released per X. laevis egg upon fertilization or activation with 10 μM ionomycin as detected by FluoZin-3 fluorometry. (E-G) Proportion of inseminated eggs that developed cleavage furrows in indicated concentrations of ZnSO4, ZnCl2, or MgSO4. Plots in (A) and (B) were fit with sigmoidal functions. (H) Proportion of development of eggs subjected to a 15-minute pretreatment with 0 or 300 μM ZnSO4 (solution 1) then washed and moved to solution (2) with 0 or 300 μM TPEN for sperm addition (N = 68–259 eggs, 5 trials). (I) Incidence of cleavage furrow development from eggs inseminated in 0 or 1 mM ZnSO4 and then transferred to a new solution with 0 or 1 mM ZnSO4 30 minutes after sperm addition (N = 52–258 eggs in 6 trials). (J) Proportion of cleavage furrow development from eggs inseminated in and transferred to control conditions (black), eggs inseminated in 300 μM ZnSO4 and transferred to 600 μM TPEN 30 minutes following sperm addition (purple), or eggs inseminated in and transferred to 1 mM ZnSO4 (red) (N = 26–58 eggs in 7 trials). (E-J) Errors are SEM. For full source data, see S1 Data. TPEN, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine.
	Fig 2. Zinc release and protection of eggs is shared in external fertilizers. (A) Representative FluoZin-3 and bright-field images of A. mexicanum oocytes before and after activation with 300 μM ionomycin (N = 6 oocytes, 2 trials). (B) Representative images of FluoZin-3 and bright-field of D. rerio eggs releasing zinc upon activation with water (N = 7 eggs, 4 trials). Eggs were in 50 μM FluoZin-3 (A and B). (C) Appearance of blastodisc and abortive cleavages confirmed activation for D. rerio eggs. (D) Box plot of zinc released per D. rerio egg upon activation as detected by fluorometry using FluoZin-3 (N = 59–138 eggs, 5 trials). Dashed lines denote the 25th and 75th percentile of the data distribution for zinc released from X. laevis eggs following fertilization. (E) Representative images of S. purpuratus eggs and embryos. (F) Development was blocked in a concentration-dependent manner for S. purpuratus eggs in ZnSO4 (N = 120–684 eggs, 5 trials). (G) Representative images of H. symbiolongicarpus unfertilized and divided eggs. (H) Development was blocked in a concentration-dependent manner for H. symbiolongicarpus eggs inseminated in extracellular ZnSO4 (N = 143–1463 eggs, 5–6 trials). Errors are SEM. For full source data, see S1 Data.
	Fig 3. Zinc inhibits embryonic development by binding to proteins surrounding the egg. (A) Incidence of development of eggs pretreated in 300 μM ZnSO4 prior to insemination (N = 34–150 eggs, 5 trials). (B) X. laevis eggs before and after jelly removal. (C) Incidence of development of jelly-free eggs in varying ZnSO4 concentrations (N = 53–96 eggs, 6 trials). (D) Average proportion of cleavage furrow development from jellied eggs pretreated in solution (1), with either 0 or 300 μM ZnSO4. After a 15-minute treatment, eggs were then washed and moved to solution (2) for sperm addition with 0 or 300 μM TPEN (N = 35–192, 5 trials). (E) Box plot distributions of the IC50 values of inhibition of appearance of cleavage furrows in X. laevis eggs inseminated in cobalt (blue), nickel (green), copper (orange), or zinc (red). (F) Proportion of division of X. laevis eggs inseminated in varying concentrations of extracellular cobalt (N = 148–295 eggs, 5 trials), nickel (N = 136–283 eggs, 5 trials), or copper (N = 230–321 eggs, 5 trials). (G) Incidence of cleavage furrow development from eggs inseminated in solution (1), with 0 or 100 μM CuCl2, and then transferred to solution (2), with 0 or 100 μM CuCl2, 30 minutes after sperm addition (N = 205–302 eggs, 5 trials). (H) Fertilization stimulates the release of zinc from the egg to modify the envelope and jelly coat to inhibit sperm entry in X. laevis. All errors are SEM. For full source data, see S1 Data. IC50, half-maximal inhibitory concentration; TPEN, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine.
	S1 Fig. Activation in the absence of fertilization promotes zinc release from X. laevis eggs and oocytes. (A) Kinetics of zinc release in activation of X. laevis and A. mexicanum eggs and oocytes. (B) Parthenogenic activation of X. laevis eggs (N = 9 eggs, 5 trials) or in vitro matured oocytes (E; N = 9 eggs, 3 trials) with 10 μM ionomycin in the presence of FluoZin-3 also induced zinc exocytosis. Red arrowheads highlight the lifting of the fertilization envelope (B, F). (C) Changes in FluoZin-3 fluorescence upon parthenogenic activation were detected by region of interest analysis. Integrated fluorescence relative to time of ionomycin addition, detected by region of interest analysis (indicated by colored boxes in upper left image). (D) Representative images of immature and in vitro matured oocytes. (F) Treatment of X. laevis eggs in FlouZin-3 with the zinc chelator TPEN abolished fertilization-induced zinc release (N = 8 eggs, 2 trials). For full source data, see S1 Data. https://doi.org/10.1371/journal.pbio.3000811.s001
	S2 Fig. Activation of immature X. laevis eggs. Zinc released upon activation of immature X. laevis oocytes with 200 μM ionomycin (N = 15 eggs, 7 trials). https://doi.org/10.1371/journal.pbio.3000811.s002

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