Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes

BACKGROUND: Mammalian oocyte activation occurs via a series of intracellular calcium (Ca2+) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLC zeta). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLC zeta gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLC zeta residue 398 (PLC zeta(H398P)), leading to abnormal Ca2+ release profiles and reduced oocyte activation efficiency. METHODS AND RESULTS: In the present study, we used HEK293T cells to produce recombinant human wild-type PLC zeta (PLC zeta(WT)) protein which, upon microinjection into mouse oocytes, induced Ca2+ oscillations characteristic of oocyte activation. Injection of recombinant PLC zeta H398P was unable to elicit Ca2+ oscillations in mouse oocytes. Loss of activity mutations, such as PLC zeta(H398P) and an artificially induced frameshift mutation (PLC zeta DYC2) did not affect Ca2+ release when over-expressed in HEK293T cells, whereas PLC zeta(WT) inhibited adenosine triphosphate-activated Ca2+ release. Confocal imaging of fluorescently tagged PLC zeta isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLC zeta(WT). PLC zeta(H398P). PLC zeta Delta(YC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLC zeta immunofluorescence from the patient exhibiting PLC zeta(H398P) compared with fertile controls. CONCLUSIONS: We demonstrate, for the first time, the production of active recombinant human PLC zeta protein which retained the ability to elicit characteristic Ca2+ oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLC zeta, and provide a critical step forward in obtaining purified PLC zeta protein as a potential therapeutic agent for oocyte activation deficiency.