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Brief history of the fast block to polyspermy

Fertilization by more than one sperm, a condition known as polyspermy, can cause gross chromosomal abnormalities and is embryonic lethal in monospermic animals. External fertilizers have adapted an immediate mechanism to prevent supernumerary fertilizations of the nascent zygote. The fast block to polyspermy is marked by a depolarization of the egg membrane where the negative resting potential rapidly changes to a positive potential at fertilization. Sperm can bind to, but no longer enter, a depolarized egg membrane.

The study of polyspermy blocking mechanisms began over 100 years ago when it was observed the sand dollar egg membrane lifted to cause a physical barrier about 30 seconds after sperm was added, and this lifting prevented additional sperm from entering the fertilized egg. In 1922, it was suggested that there was an electrical component to maintaining fertilization by one sperm. The idea that there was an electrical barrier preventing fertilization was not actually observed until almost 30 years later when Tyler et al. used a starfish egg and demonstrated that there is a depolarization of the egg membrane at fertilization through the first successful microelectrode recording of the membrane. In 1976, Laurinda Jaffe successfully utilized sea urchin eggs to show that the depolarization of the membrane was responsible for keeping additional sperm out of the egg by holding the egg at different voltages and observing sperm entrance. When eggs were held at a negative membrane potential, multiple sperm entered the egg. However, when the eggs were held at a positive potential, the egg was not fertilized.

The fast block to polyspermy in Xenopus laevis

Perhaps one of the most well studied cases of the fast block to polyspermy is in the African clawed frog, Xenopus laevis. X. laevis eggs express TMEM16A, which is a Ca2+-activated Cl- channel. Upon activation following fertilization, TMEM16A releases an efflux of Cl- ions that causes the egg membrane to depolarize to prevent further sperm entry. The resting potential of X. laevis eggs prior to fertilization is typically anywhere from -20 to -10 mV which allows sperm to bind and fuse to the egg, and the fertilization potential is typically 0 to +20 mV, which is a range of potentials that stops sperm from entering the egg. The Ca2+ required to activate TMEM16A is released from the endoplasmic reticulum when inositol trisphosphate (IP3) binds the IP3 receptor. Previous research revealed that inhibiting the IP3R in X. laevis eggs eliminated the fertilization-evoked depolarization, causing polyspermic fertilization. The production of IP3 in the egg is the result of phospholipase C (PLC) cleaving PIP2 to form diacylglycerol (DAG), which remains in the plasma membrane, and IP3. When PLC is inhibited in X. laevis eggs, the fast block depolarization was abolished and polyspermic fertilization took place,.

The method for how fertilization activates the PLC required for the fast block is an ongoing area of research. Through analysis of RNA sequencing and proteomics datasets, it has been determined that there are three PLC subtypes in mature, fertilization-competent X. laevis eggs – PLCγ1, PLCβ1, and PLCβ3,. PLC isoforms have different methods of canonical activation. PLCγ is typically activated through tyrosine phosphorylation of a specific residue via tyrosine kinase activation. PLCβ is canonically activated through the Gα subunit of heterotrimeric G-proteins, activated by G protein-coupled receptors. It has been found that inhibiting these canonical methods of activation for the three PLC subtypes in X. laevis eggs does not prevent the occurrence of the fast block to polyspermy depolarization, and monospermic fertilization is observed. This research supports that the PLC subtypes are not activated through well-established mechanisms, and a novel mechanism is responsible for signaling the activation of PLC during the fast block following sperm-egg interaction at fertilization.