Draft:The Fast Block to Polyspermy in the African clawed frog, Xenopus laevis

• Comment: Reads more like a college assignment or journal article than an encyclopedia article. I dream of horses ^{(Contribs) (Talk)} 17:04, 2 April 2023 (UTC)

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Preventing polyspermy during fertilization

In most animals, the fertilization of an egg by more than one sperm leads to severe chromosomal problems that prevent embryonic development. To prevent this from happening, eggs have evolved various mechanisms to prevent additional sperm from entering after the initial fertilization. These protective mechanisms are known as polyspermy blocks, with the two most common types being the fast block and the slow block. For animals that reproduce externally (outside the mother's body), fertilization triggers a change in the egg's cell membrane called depolarization in which the electrical charge in the egg becomes less negative. Sperm can attach, but not enter, a depolarized egg. Thus depolarization prevents more than one sperm from fertilizing the egg.

Discovery of the polyspermy block

The concepts of fast and slow blocks to polyspermy were first proposed over a 100 years ago. In the late 1910's, the biologist Ernest E. Just observed that the addition of sperm to sand dollar eggs induced the formation of a shell-like structure within approximately 30 seconds. This "shell" functioned to prevent additional sperm from entering the fertilized egg, hence a "slow block" to polyspermy. E.E. Just speculated that since numerous sperm reached the egg within this short, 30 second time frame, a more immediate barrier was likely necessary to stop polyspermy. 1922, J. Gray hypothesized that an electrical change in the egg ensured that only a single sperm fertilized the egg. This concept gained support when, three decades later, Tyler and colleagues observed that the egg membrane in starfish eggs depolarized following fertilization. The significance of this depolarization in preventing polyspermy became clearer in 1976 when Laurinda Jaffe discovered that maintaining a depolarized state in sea urchin eggs prevented sperm from entering. In contrast, creating a hyperpolarized state in sea urchin eggs allowed multiple sperm to enter the egg (polyspermy). The finding from Jaffe established that the "fast block" to polyspermy is triggered by the fertilization-induced depolarization. Since these seminal experiments, researchers have identified fast blocks to polyspermy in other externally reproducing animals, including frogs.

Fast block to polyspermy in Xenopus laevis

The African clawed frog, Xenopus laevis or X. laevis, is an externally fertilizing animal that uses a depolarization-mediated fast block to polyspermy. Depolarization in X. laevis eggs is driven by an increase in cytosolic calcium ions (Ca²⁺) and efflux (flowing out) of chloride ions (Cl^-). The ion channel that produces the depolarizing current is the transmembrane protein TMEM16A (ANO1 in humans). TMEM16A is a Ca²⁺ -activated Cl^- channel: cytosolic Ca²⁺ binds to TMEM16A to activate it and promote Cl- efflux, causing the egg membrane to depolarize. This depolarization effectively stops additional sperm from entering the egg after fertilization. To activate TMEM16A, Ca²⁺ ions are released from the endoplasmic reticulum when the signaling molecule inositol trisphosphate (IP₃) binds to the IP₃ receptor. Inhibiting the IP₃ receptor in X. laevis eggs prevents depolarization induced by fertilization, causing polyspermic fertilization.

IP₃ is produced when the enzyme phospholipase C (PLC) splits phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol (DAG) and IP₃. X. laevis eggs express three PLC isotypes – PLCγ1, PLCβ1, and PLCβ3. PLC isotypes have different mechanisms of canonical activation. PLCγ is typically activated through tyrosine phosphorylation of a specific residue (Y776 in X. laevis and Y783 in mice) via tyrosine kinase activation. In contrast, PLCβ is typically activated by the Gα subunit of heterotrimeric G-proteins that function downstream of G protein-coupled receptors.

PLC regulation and function in depolarization

Just how PLCs regulate fertilization in X. laevis is not clear. Tyrosine kinase inhibitors such as lavendustin A or tyrphostin B46 that target (and block) PLCγ halt calcium waves during fertilization. Likewise, blocking tyrosine phosphorylation with genistein halts X. laevis post-fertilization egg development. Finally, fertilization activates PLCγ via tyrosine phosphorylation and PLCγ is required to increase calcium levels following fertilization. These findings support a model in which sperm attachment to the egg cell membrane initiates a singling cascade — possibly through uroplakin III and Src family kinase — that leads to tyrosine phosphorylation and activation of PLCγ to initiate Ca²⁺ release and depolarization.

However, other results support an alternative model. For example, inhibiting PLCγ function using a dominant negative strategy or blocking activation of PLCβ with function blocking antibody did not prevent an increase in calcium levels post-fertilization, suggesting the PLC activation/function is not necessary for depolarization in X . laevis eggs. Likewise, more recent experiments that inhibited all three PLC isotypes using the tyrosine kinase inhibitors genistein and dasantinib and the G-protein inhibitor YM-254890 found that blocking PLC activity did not prevent the fast block to polyspermy depolarization. Instead, eggs showed monospermic fertilization in the presence of the inhibitors. These findings suggest that the canonical PLC activation pathways do not function during fertilization-induced depolarization. Instead, a yet undefined mechanism could regulate PLC activation during the fast block.