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spermatozoa, which, after injected into eggs, is able to fully replicate the Ca2+
fingerprint of the sperm. The Ca2+ elevation in newt eggs is also stimulated by the
microinjection of porcine citrate synthase and by the injection of citrate synthase
mRNA as well (Harada et al. 2007 ). Inhibition of the enzyme’s activity prevents
egg activation triggered not only by a crude sperm extract but also by the fertilizing
sperm (Harada et al. 2011 ). It seems that citrate synthase elevates PLC activity in
the egg through a yet unknown mechanism. The enzyme normally produces citrate
from acetyl-CoA and oxaloacetate in the mitochondrial tricarboxylic acid (TCA)
cycle, but it can also inversely cleave citrate into acetyl-CoA and oxaloacetate,
molecules that are able to activate newt eggs. Although it is not clear how these
citrate-derived products trigger the Ca2+ signal at fertilization, in other cell types,
acetyl-CoA has been shown to sensitize IP 3 receptors (Missiaen et al. 1997 ), and
oxaloacetate is able to stimulate Ca2+ release from mitochondria (Leikin et al.
1993 ). Most citrate synthase is localized in the neck and midpiece region of the
sperm cell, outside the mitochondria. Because all sperm components, including the
tail, are incorporated into the egg at fertilization, citrate synthase is exposed to the
ooplasm soon after sperm-egg fusion (Iwao 2012 ).
Another proposed sperm factor has been tr-kit, a truncated form of the c-kit
receptor, which is known to play an important role in primordial germ cell migra-
tion (Sette et al. 1997 ). In mouse eggs, tr-kit causes activation, supposedly by
stimulating the enzyme PLCγ1 through a Src-like kinase, Fyn (Sette et al. 2002 ).
Tr-kit-induced egg activation is blocked by a PLCγ SH3 construct, but interest-
ingly, the same SH3 construct does not interfere with fertilization (Mehlmann
et al. 1998 ). In addition, tr-kit has never been shown to induce repetitive Ca2+
transients in mammalian eggs, which is an expected characteristic of a bona fide
sperm factor. Finally, the postacrosomal sheath WW domain-binding protein
(PAWP) has also been listed as a candidate sperm factor (Wu et al. 2007 ). The
protein resides in the postacrosomal sheath subcompartment of the sperm perinu-
clear theca (a localization consistent with that of a potential sperm factor), and it is
released into the ooplasm at fertilization. Microinjection of recombinant PAWP
into mature frog and porcine eggs stimulates Ca2+ release and egg activation (Wu
et al. 2007 ; Aarabi et al. 2010 ), and the recombinant protein or its complementary
RNA (cRNA) is also able to elicit Ca2+ oscillations and pronuclear formation in
human and mouse eggs (Aarabi et al. 2014 ). In addition, the Ca2+ transients can be
blocked by co-injection of an inhibitory peptide derived from the WW domain-
binding motif of PAWP, and this same peptide is also able to inhibit sperm-induced
Ca2+ oscillations suggesting that PAWP has a role in egg activation during fertiliza-
tion. However, it is unclear what pathway PAWP might use to generate the Ca2+
signal, and its ability to induce Ca2+ oscillations could not be confirmed by other
groups (Nomikos et al. 2014 , 2015 ). Recently, PAWP null mice have been pro-
duced, and although the sperm of these animals lacked PAWP protein, they were
able to fertilize eggs and induce Ca2+ oscillations (Satouh et al. 2015 ). Thus, the
recognition of PAWP as a true sperm-borne oocyte-activating factor requires fur-
ther verification.
Z. Machaty et al.