23ability to trigger regenerative Ca2+ oscillations (Parrington et al. 2002 ). These data
suggested that the sperm factor might be a novel PLC isoform.
The quest for new PLC varieties led to the identification of a set of sequences in a
mouse expressed sequence tag (EST) database; this was followed by the amplifica-
tion of a novel PLC from a mouse spermatid cDNA library (Saunders et al. 2002 ).
The new isoform was named PLCζ (PLCzeta). It was found to be smaller than all
other known mammalian PLC isoforms, and Northern blot analysis revealed that it
was expressed exclusively in the testis. PLCζ orthologues have also been identified in
the sperm of other mammalian species such as hamster, pig, horse, monkey, and
human (reviewed by Nomikos et al. 2013 ). Complementary RNA (cRNA) of the
mouse, cynomolgus monkey, and human PLCζ or the recombinant protein is able to
recapitulate the sperm-induced Ca2+ oscillations in mouse eggs (Saunders et al. 2002 ;
Cox et al. 2002 ; Kouchi et al. 2004 ). In human and pig eggs, the PLCζ cRNA triggers
embryo development to the blastocyst stage (Rogers et al. 2004 ; Yoneda et al. 2006 ),
and immunodepletion of sperm extracts using an anti-PLCζ antibody abolishes the
extract’s ability to induce the oscillations (Saunders et al. 2002 ). PLCζ protein in
mouse sperm resides in the postacrosomal region of the perinuclear theca, a con-
densed layer of cytosolic proteins that covers the nucleus (Young et al. 2009 ); in bull
sperm it localizes in the equatorial region (Yoon and Fissore 2007 ). This localization
is consistent with the expectation that the Ca2+ release-inducing factor must gain rapid
access to the egg cytoplasm after gamete fusion (Lawrence et al. 1997 ). Approximately
40 fg PLCζ in the ooplasm is sufficient to induce Ca2+ oscillations; this amount cor-
relates with the range of PLCζ estimated to be present in a single mouse sperm
(Saunders et al. 2002 ). Finally, transgenic mice have been created that showed
reduced expression of PLCζ; when spermatozoa of such animals were used for
in vitro fertilization, the induced Ca2+ oscillations terminated prematurely in the eggs
(Knott et al. 2005 ). PLCζ-deficient male mice have also been generated (Ito et al.
2010 ); unfortunately, such animals fail to make sperm so the ultimate experiment to
demonstrate the critical role of PLCζ during fertilization is yet to be conducted.
Nevertheless, the data listed above strongly support the notion that mammalian sperm
generate the Ca2+ signal that triggers embryo development by means of PLCζ.
The PLCζ enzymes identified in different mammals are all similar in size (Swann
et al. 2006 ). Interestingly, the enzyme lacks an N-terminal pleckstrin homology (PH)
domain that is present in other PLC isoforms and instead contains two pairs of EF
hand domains at the N-terminus, followed by the X-Y catalytic domain found in all
mammalian PLC enzymes and a PKC-homology type II (C2) domain at its
C-terminus. The X-Y catalytic domain is the most highly conserved region of the
molecule and is responsible for its enzymatic activity. A point mutation in this domain
leads to a complete loss of the enzyme’s ability to hydrolyze PIP 2 in vitro and, hence,
to induce Ca2+ oscillations in mouse eggs (Nomikos et al. 2011 ). The EF hands pos-
sess Ca2+-binding residues similar to those found in other Ca2+-binding proteins
(Kouchi et al. 2005 ). They confer the enzyme high Ca2+ sensitivity: PLCζ is 100-fold
more sensitive to Ca2+ than PLCδ1, the isoform it shares the greatest homology with
(Nomikos et al. 2005 ). PH domains can bind phosphoinositides in cellular mem-
branes; most proteins with a PH domain need to associate with the membrane to
1 Egg Activation at Fertilization