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 (PLCζ ^H398P, histidine > proline) and PLCζ ^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 PLCζ ^H398P and PLCζ ^H233L, and investigation of localization patterns arising from the expression of fluorescently tagged PLCζ isoforms in HEK293T cells. Main Outcome Measure(s): The presence/absence of PLCζ ^H398P and PLCζ ^H233L determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLCζ isoforms quantified in HEK293T cells. Result(s): Sperm possessed either PLCζ ^H233L or PLCζ ^H398P, but never both at the same time. Fluorescent PLCζ ^H233L and PLCζ ^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 PLCζ ^WT > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLCζ ^H233L+H398P exhibited a statistically significantly reduced level of fluorescence compared with PLCζ ^H398P at 48 hours but not 56 hours. Conclusion(s): Both H398P and H233L mutations are present on different alleles and do not alter PLCζ localization in HEK293T cells. Loss-of-activity mutations in PLCζ may contribute not only toward male infertility but also male subfertility in cases where PLCζ is mutated on a single allele.