25the Ca2+ signal during mammalian fertilization. Intracytoplasmic sperm injection
(ICSI) is a powerful technique used in assisted reproduction to improve conditions
of male infertility. It has been found that sperm of a number of patients, which
repeatedly failed to stimulate embryo development after ICSI, were unable to trigger
Ca2+ oscillations and showed reduced or complete absence of PLCζ expression
(Yoon et al. 2008 ; Heytens et al. 2009 ). In addition, patients with a history of failed
ICSI were shown to have a point mutation in the X or Y domain of the PLCζ gene
(Kashir et al. 2012 ). Injection of spermatozoa from these patients or cRNA encod-
ing the mutant PLCζ was unable to induce Ca2+ oscillation in mouse eggs indicating
the pivotal role of PLCζ in the generation of the Ca2+ signal.
The situation in other vertebrate groups is far less clear. Supernatants of fish
sperm homogenates trigger activation in medaka eggs after microinjection indicat-
ing the presence of an egg-activating factor (Iwamatsu and Ota 1974 ). Sperm
extracts from tilapia (Coward et al. 2003 ) and recombinant medaka PLCζ (Ito et al.
2008 ) induce repetitive Ca2+ transients after injection into mouse eggs.
Intracytoplasmic injection of sperm into eggs leads to normal fertilization and the
formation of healthy embryos in medaka suggesting that the fertilization Ca2+ signal
is triggered by a diffusible factor in the sperm rather than the stimulation of a sur-
face receptor located on the egg (Otani et al. 2009 ). Similarly, extracts prepared
from chicken sperm induce repetitive Ca2+ transients after injection into mouse eggs
(Dong et al. 2000 ), and recombinant chicken PLCζ has the same effect (Coward
et al. 2005 ). Also, the developmental potential of quail embryos produced by intra-
cytoplasmic sperm injection improves when PLCζ is co-injected into the eggs
(Mizushima et al. 2008 ), and microinjection of extracts from newt sperm causes a
wavelike Ca2+ increase in newt eggs (Yamamoto et al. 2001 ). These findings seem
to support the sperm factor model in nonmammalian vertebrates; whether the factor
is used to stimulate egg activation under physiological conditions is a different
question. Xenopus sperm extracts cause regenerative Ca2+ rises in mouse eggs
(Dong et al. 2000 ), but interestingly, they are unable to induce activation in Xenopus
eggs (Harada et al. 2011 ). In addition, Xenopus eggs are insensitive to newt sperm
extracts as well (Harada et al. 2011 ) indicating that these eggs do not contain the
signaling cascade required for stimulation by a diffusible sperm factor, at least those
present in Xenopus and newt spermatozoa. This finding seems to be consistent with
the observations that in Xenopus, egg activation is triggered as a result of membrane
interactions between the gametes.
A number of additional molecules have also been proposed to serve as a sperm-
resident activating factor in vertebrate species. Isoforms of PLCζ were found to be
present in pufferfish (Coward et al. 2011 ); unexpectedly, it is expressed in the
ovary and brain instead of the testis. Injection of its cRNA does not cause Ca2+
oscillations in mouse eggs potentially indicating that pufferfish PLCζ has no role
in Ca2+ mobilization. Alternatively, this may also suggest that in the pufferfish
PLCζ is expressed in the egg rather than the sperm and it needs interaction with
other molecules localized in the male or female gamete to become capable of induc-
ing Ca2+ release. In the newt, a 45-kDa protein termed citrate synthase is present in
1 Egg Activation at Fertilization