To test if PLCζ was the sole calcium trigger in sperm, Fissore’s team created a PLCζ-null mouse using CRISPR/Cas9. Using CRISPR, they targeted the Plcz1 gene in fertilized mouse eggs. They came up with two strategies as to go about this: Cas9WT with a single sgRNA and Cas9D10A nickase with paired sgRNAs. A line of sperm where Cas9D10A induced a 22 nucleotide deletion in the target area (Plcz1em1Jparr), and another where Cas9WT induced a 17 nucleotide deletion in another area (Plczl em2Jparr) were created. These deletions created two mutant alleles for the Plcz1 gene. When injected into mouse eggs, the mutant cRNA did not induce calcium oscillations confirming that the mutants were PLCζ-null (Plcz1-/-).
They found that the loss of PLCζ did not affect the sperms quality or its ability to bind and fuse with an egg. Wild-type sperm (Plcz1+) and Plcz1-/- sperm (Plcz1-) response to progesterone or ionoycin were undistinguishable from each other. When testing the Plcz1+ sperm in wild-type mouse eggs, the sperm induced calcium oscillations “symptomatic of those seen at fertilization in nearly all eggs.” The Plcz1- did not. This was then confirmed with using in vitro fertilization of zone pellucid-free eggs (eggs without a glycoprotein layer around its plasma membrane). 16 out of 34 eggs with Plcz1+ sperm showed Ca2+ oscillations and none of the eggs with Plcz1 em1Jparr derived sperm show any oscillations. His team showed the first direct evidence that PLCζ is the “sole physiological trigger of the Ca2+ oscillations responsible for egg activation in mammals.”
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