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

Sperm-specific phospholipase C zeta (PLC zeta) 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 PLC zeta C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLC zeta. Microinjection of cRNA or recombinant protein corresponding to PLC zeta(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 PLC zeta (I489F) protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLC zeta(I489F) mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3) P and PI(5) P-containing liposomes compared with wild-type PLC zeta. Our findings highlight the importance of PLC zeta at fertilisation and the vital role of the C2 domain in PLC zeta function, possibly due to its novel binding characteristics.