TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs

Abstract

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+ activated Cl- channel TMEM16A. To do so, fertilization activates a phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLC{gamma} and PLC{beta}. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLC{gamma}, or inhibitors of Gq/11 pathways, used to stop activation of PLC{beta}. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.