Extracellular Zinc Acts as a Dynamic Extracellular Regulator of Fertilization in Xenopus laevis

For most animals, fertilization of an egg by more than one sperm is embryonically lethal, and eggs therefore deploy multiple blocks to prevent additional sperm from entering an already fertilized egg. While Zn2+ release has emerged as a conserved feature of the slow block, its precise role in external fertilizers remains unclear. Here, we demonstrate that Xenopus laevis fertilization triggers a robust efflux of Zn2+ and that insemination of X. laevis eggs in physiologically relevant concentrations of Zn2+ potently and reversibly suppresses early development. Our findings are consistent with the hypothesis that extracellular Zn2+ inhibits fertilization through multiple mechanisms. Extracellular Zn2+ prevents the fertilization-evoked depolarization, also called the fast block to polyspermy, by acting upstream of the Ca2+-activated Cl- channel TMEM16A. Zn2+ also directly reduces sperm fertilizing capacity, although eggs are significantly more sensitive than sperm. Furthermore, Zn2+ coordinates with the egg's extracellular matrix to stabilize the glycoprotein-rich jelly coat, rendering it resistant to reductive removal and suggesting a structural mechanism by which Zn2+ limits sperm entry. The inhibitory effects of Zn2+ are fully reversed by the extracellular chelator ZX1, reinforcing the conclusion that Zn2+ functions as a dynamic extracellular regulator rather than an intracellular messenger. Our findings establish Zn2+ as a multi-level signal that reinforces fertilization barriers by targeting both gametes and modifying the egg's extracellular environment.