Zinc & Fertilization

An explainer for our 2020 PLOS Biology manuscript

In 2010, the Woodruff and O’Halloron labs demonstrated that eggs release zinc following fertilization in mouse eggs. Slowly after sperm entry, eggs release cortical granules, vesicles docked at the egg membrane, via exocytosis. Contents of the cortical granules include enzymes that modify the zona pellucida to stop additional sperm from entering an already fertilized egg, in a process referred to as the slow block to polyspermy. Evidently, these cortical granules are enriched in zinc. Because zinc is released during the slow block, we speculated that zinc contributed to protection of the nascent zygote to supernumerary fertilizations. Because Xenopus laevis is an excellent system for fertilization studies, we began to explore whether zinc was also released from frog eggs at fertilization. To test this hypothesis, we applied sperm to X. laevis eggs in the presence of the fluorescent zinc indicator FluoZin-3. Indeed, we observed that zinc was released in a slow wave that wrapped around the egg minutes after sperm application. This zinc release coincided with the lifting of the vitelline envelope (the extracellular structure that homologous to the zona pellucida).

We next wondered if zinc contributes to the slow block. We reasoned that if extracellular zinc stopped sperm entry into already fertilized eggs, that insemination in the presence of extracellular zinc should stop fertilization altogether. Using the appearance of cleavage furrows as an indicator of successful fertilization, we observed that increasing concentrations of zinc stopped development. Moreover, no cleavage furrows were observed in eggs pre-treated with zinc but inseminated in the absence of excess zinc. This zinc inhibition was reversible with the chelator TPEN. To home in on the time frame of when zinc interferes with fertilization and early embryonic development, we measured the zinc induced shift in the appearance of cleavage furrows. Under control conditions, cleavage furrows appeared in half of the zygotes 77 min after sperm addition. By contrast, when we inseminated eggs in the presence of zinc, then 30 min after sperm addition, we applied the chelator TPEN, cleavage furrows appeared in half of the zygotes at 111 minutes. This 34 min shift in cleavage furrow development was near the 30 min between insemination and TPEN addition.  Altogether, these data suggest that zinc contributes to the slow block.

We next considered whether zinc was released during activation of eggs from other animals. We started with the neotenic salamander axolotl (Ambystoma mexicanum). Again, using FluoZin-3 we observed that these eggs released extracellular zinc with activation with activation by calcium ionophore. Zebrafish eggs are activated by hydration rather than insemination. We also observed zinc release from these eggs.

We next turned to two invertebrate animals: the sea urchin (Strongylocentrotus purpuratus) and the hydroid Hydractinia symbiolongicarpus. Both of these animals fertilize in the ocean, in a process that requires a high concentration of extracellular calcium. The requirement of millimolar calcium prevented us from using FluoZin-3 to observed extracellular zinc. So, we turned to our functional assay. By inseminating in the presence of increasing concentrations of extracellular zinc, we observed that cleavage furrow development was inhibited in a concentration dependent manner.

These data suggest that zinc can contribute to the protection of zygotes from multiple fertilizations in a process conserved from Cnidaria to mammals.