Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ

Abstract

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca²⁺) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the <i>in vivo</i> Ca²⁺ oscillation-inducing activity and the <i>in vitro</i> biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζ^I489F mutant at physiological concentrations completely failed to cause Ca²⁺ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζ^I489F protein, leading to Ca²⁺ oscillations and egg activation. Our <i>in vitro</i> biochemical analysis suggested that the PLCζ^I489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.