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

Mammalian oocyte activation occurs via a series of intracellular calcium (Ca2+) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). 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 PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζH398P), leading to abnormal Ca2+ release profiles and reduced oocyte activation efficiency. In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζWT) protein which, upon microinjection into mouse oocytes, induced Ca2+ oscillations characteristic of oocyte activation. Injection of recombinant PLCζH398P was unable to elicit Ca2+ oscillations in mouse oocytes. Loss of activity mutations, such as PLCζH398P and an artificially induced frameshift mutation (PLCζΔYC2) did not affect Ca2+ release when over-expressed in HEK293T cells, whereas PLCζWT inhibited adenosine triphosphate-activated Ca2+ release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζWT > PLCζH398P > PLCζΔYC2, 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 PLCζ immunofluorescence from the patient exhibiting PLCζH398P compared with fertile controls. We demonstrate, for the first time, the production of active recombinant human PLCζ 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 PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency.