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.


METHODS
Laboratory-based study.


METHODS
University laboratory.


METHODS
An infertile 38-year-old man with significantly impaired oocyte activation ability.


METHODS
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Nuffield Department of Obstetrics and Gynaecology, Women's Centre, John Radcliffe Hospital, Headington, Oxford, United Kingdom.
Junaid
J
Kashir
Michalis
M
Konstantinidis
Celine
C
Jones
Bjorn
B
Heindryckx
Petra
P
De Sutter
John
J
Parrington
Dagan
D
Wells
Kevin
K
Coward
D000328:Adult; D000483:Alleles; D015971:Gene Expression Regulation, Enzymologic; D006580:Genetic Carrier Screening; D057809:HEK293 Cells; D006801:Humans; D007248:Infertility, Male / Q000201:enzymology / Q000235:genetics*; D008297:Male; D009154:Mutation / Q000235:genetics*; D009865:Oocytes / Q000201:enzymology / Q000502:physiology*; D054801:Phosphoinositide Phospholipase C / Q000096:biosynthesis / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D013094:Spermatozoa / Q000201:enzymology / Q000502:physiology*
C499235:PLCZ1 protein, human; D054801:Phosphoinositide Phospholipase C