Characterization of two heterozygous mutations of the oocyte activation factor phospholipase C zeta (PLCζ) from an infertile man by use of minisequencing of individual sperm and expression in somatic cells

Objective: To examine the underlying factors leading to infertility in a male patient from whom phospholipase C zeta H398P (PLC zeta(H398P), histidine > proline) and PLC zeta(H233L) (histidine > leucine) mutations were previously identified. Design: Laboratory-based study. Setting: University laboratory. Patient(s): An infertile 38-year-old man with significantly impaired oocyte activation ability. Intervention(s): Minisequencing of individual sperm for PLC zeta(H398P) and PLC zeta(H233L), and investigation of localization patterns arising from the expression of fluorescently tagged PLC zeta isoforms in HEK293T cells. Main Outcome Measure(s): The presence/absence of PLC zeta(H398P) and PLC zeta(H233L) determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLC zeta isoforms quantified in HEK293T cells. Result(s): Sperm possessed either PLC zeta(H233L) or PLC zeta(H398P), but never both at the same time. Fluorescent PLC zeta(H233L) and PLC zeta(H233L+H398P) (both mutations together) localized to discrete regions in HEK293T cytoplasm but not the plasma membrane. Fluorescence statistically significantly varied between constructs such that PLC zeta(WT) > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLC zeta(H233L+H398P) exhibited a statistically significantly reduced level of fluorescence compared with PLC zeta(H398P) at 48 hours but not 56 hours. Conclusion(s): Both H398P and H233L mutations are present on different alleles and do not alter PLC zeta localization in HEK293T cells. Loss-of-activity mutations in PLC zeta may contribute not only toward male infertility but also male subfertility in cases where PLC zeta is mutated on a single allele. (Fertil Steril (R) 2012;98:423-31. (C) 2012 by American Society for Reproductive Medicine.)