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

Capsule:
Investigating contributory factors to infertility in a man with two phospholipase C zeta (PLCz) mutations found loss-of-activity mutations in PLCz potentially resulting in a slower rate of mutant protein transcription.

Authors:
Junaid Kashir, Ph.D., Michalis Konstantinidis, B.Sc., Celine Jones, Bjorn Heindryckx, Ph.D., Petra De Sutter, M.D., Ph.D., John Parrington, Ph.D., Dagan Wells, Ph.D., Kevin Coward, Ph.D.

Volume 98, Issue 2 , Pages 423-431, August 2012

Abstract:

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.

  • kivan

    Very thorough study. Continued identification of additional evidence-based genetic links to infertility will eventually allow us to screen patients pre-IVF to determine the best treatment regimen and potential genetic risks to offspring.

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