24
function. The fact that PLCζ lacks such a domain is consistent with the finding that
PLCζ does not specifically localize in the oolemma following its release from the
sperm but allocates throughout the entire cytoplasm (Yoda et al. 2004 ). This implies
that the enzyme either lacks a targeting mechanism or, alternatively, is recruited to a
pool of PIP 2 that does not reside in the plasma membrane. In fact, PIP 2 in mouse eggs
seems to localize not in the plasma membrane (as it does in most other cell types) but
in vesicles in the cytoplasm. Immunocytochemical experiments have indicated that
PLCζ also accumulates in such vesicles after gamete fusion (Yu et al. 2012 ).
The C2 domain is generally found in proteins that can bind to phospholipids
(Nalefski and Falke 1996 ). Most C2 domains bind to Ca2+ (although there are some
that do not), and Ca2+ binding to the C2 domain is generally crucial for enzyme
activity (Zheng et al. 2000 ). Deletion of this domain obliterated the ability of PLCζ
to induce Ca2+ oscillations without affecting enzyme activity (Nomikos et al. 2005 ),
indicating that the C2 domain is essential for proper PLCζ function. The X-Y
linker, the segment that joins together the X and Y sections of the catalytic domain,
may also be involved in binding PLCζ to biological membranes. It was proposed
that a cluster of positively charged residues within this region might be responsible
for membrane targeting, possibly via electrostatic interactions with negatively
charged PIP 2 (Nomikos et al. 2007 ). The successive reduction of the net positive
charge within the X-Y linker (Nomikos et al. 2011a) or the deletion of the entire
X-Y linker region (Nomikos et al. 2011b) led to a significant decrease in the
enzyme’s ability to interact with PIP 2 under in vitro conditions and to induce Ca2+
oscillations. In addition, the X-Y linker shows marked differences between spe-
cies, being the shortest in humans and longest in the cynomolgus monkey (Swann
et al. 2006 ). This variation might be partially responsible for the diverse potency of
PLCζ of different species to catalyze PIP 2 hydrolysis and generate Ca2+ transients
(Saunders et al. 2007 ).
In mice, the sperm-induced Ca2+ oscillations stop around the time the male and
female pronuclei are formed, possibly because the forming pronuclei sequester
PLCζ that halts the signal. This idea is supported by the results of immunocyto-
chemical analyses indicating that recombinant mouse PLCζ accumulates in the pro-
nuclei upon the cessation of the oscillations (Larman et al. 2004 ). The observation
that the basic residues in the X-Y linker region constitute a nuclear localization
sequence is consistent with this finding (Ito et al. 2008 ). Mutational studies have
indicated that replacing the basic residues with acidic ones in the nuclear localiza-
tion signal results in a loss of the nuclear translocation ability and the oscillations do
not terminate at the time of pronuclear formation. Although putative nuclear local-
ization sequences have been predicted in the PLCζ of other species, medaka, rat,
and human PLCζ are unable to localize in mouse pronuclei (Ito et al. 2008 ). In
addition, rat PLCζ does not accumulate in rat pronuclei although the mouse PLCζ
does. This implies that unlike in mice, PLCζ of other species may not be seques-
tered by the newly formed pronuclei.
Finally, clinical reports that linked deficiency in human PLCζ to male infertility
provided further evidence to support the central role of PLCζ in the generation of
Z. Machaty et al.