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
2012
Kashir, Junaid | Konstantinidis, Michalis | Jones, Celine | Heindryckx, Bjorn | Sutter, Petra de | Parrington, John | Wells, Dagan | Coward, Kevin
OBJECTIVE: To 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. 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 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.
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