Reproduction Abstracts

Introduction: 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 and early embryonic development during mammalian fertilization. Sperm-delivered PLCζ hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP₂) stores within the ooplasm, triggering Ca²⁺ oscillations through the inositol 1,4,5-trisphosphate (IP₃) signaling pathway. PLCζ consists of four EF hand domains at the N-terminus, the characteristic X and Y catalytic domains in the centre, followed by a C-terminal C2 domain. 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 (I) residue (I489) had been substituted with a phenylalanine (F). Herein, we have analysed the effect of this mutation on the in vivo Ca²⁺ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ.

Methods: For comparative analysis, bacterially-expressedrecombinant proteins or cRNA encoding luciferase-tagged versions of wild-type and PLCζ^I489F mutant were microinjected into unfertilised mouse eggs. The enzymatic and biochemical properties of PLCζ^WT and PLCζ^I489F mutant were analysed using an in vitro [³H]PIP₂hydrolysis and liposome binding assays.

Results and discussion: Microinjection of cRNA or recombinant protein corresponding to PLCζ^I489F mutant at physiological concentrations completely failed to cause Ca²⁺oscillations in eggs. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζ^I489Fprotein, leading to Ca²⁺ oscillations and egg activation. Our in vitro 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 to wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilization and the vital role of the C2 domain in PLCζ function due to its direct interaction(s) with either PI(3)P, PI(5)P or other unidentified egg proteins.