Polyspermy Blocks used by the African Clawed Frog Xenopus laevis

Preventing polyspermy, the fertilization of an egg by multiple sperm, is essential for the normal embryonic development of most sexually reproducing species. Eggs therefore have multiple mechanisms to prevent the entry of sperm into an already fertilized egg. The two most common polyspermy prevention mechanisms are known as the fast and slow polyspermy blocks. As these names imply, the fast block occurs within seconds of fertilization to create an electrical barrier to inhibit entry of additional sperm. The fast block has only been observed in externally fertilizing organisms, such as frogs and sea urchins, where the sperm-to-egg ratio is elevated at the moment of fertilization. The pathway by which sperm-egg contact culminates in membrane depolarization was not known for any species. I studied the fast polyspermy block in the African clawed frog, Xenopus laevis. Using electrophysiology, developmental biology, and bioinformatics techniques, I uncovered that the Ca2+-activated Cl- channel, TMEM16A, conducts the depolarizing current of the fast block, and that fertilization opens this channel in a signaling pathway requiring phospholipase C and inositol triphosphate. In a separate line of experimentation, I studied the slow polyspermy block in X. laevis; a pathway that occurs ubiquitously in sexual reproducers within minutes of fertilization. In this pathway, eggs exocytose cortical granules to release compounds that transform the extracellular matrix that surrounds eggs into a protective barrier impenetrable by sperm. The mechanisms enabling the creation of this barrier were largely-unknown. Using confocal microscopy and fluorometry, I determined that fertilization induces the release of Zn2+ from cortical granules in frogs and fish eggs, similar to mammalian eggs. Furthermore, exposing frog and sea urchin eggs to extracellular Zn2+ hindered fertilization in a concentration-dependent manner. Taken together, these results suggesting that fertilization-induced Zn2+ release is a conserved process that may contribute to the slow polyspermy block. Overall, my work aims to broaden our understanding of the diverse pathways that block polyspermy