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

ObjectiveTo examine the underlying factors leading to infertility in a male patient from whom phospholipase C zeta H398P (PLCζH398P, histidine > proline) and PLCζH233L (histidine > leucine) mutations were previously identified.DesignLaboratory-based study.SettingUniversity laboratory.Patient(s)An infertile 38-year-old man with significantly impaired oocyte activation ability.Intervention(s)Minisequencing of individual sperm for PLCζH398P and PLCζH233L, 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 PLCζH398P and PLCζH233L determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLCζ isoforms quantified in HEK293T cells.Result(s)Sperm possessed either PLCζH233L or PLCζH398P, but never both at the same time. Fluorescent PLCζH233L and PLCζH233L+H398P (both mutations together) localized to discrete regions in HEK293T cytoplasm but not the plasma membrane. Fluorescence statistically significantly varied between constructs such that PLCζWT > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLCζH233L+H398P exhibited a statistically significantly reduced level of fluorescence compared with PLCζH398P 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.