21domains of PLCγ inhibit PLCγ activation by the receptor, they are unable to block
Ca2+ release at fertilization (Mehlmann et al. 1998 ; Runft et al. 1999 ). Finally, when
the phosphatidylinositol signaling system is artificially activated in mammalian
eggs (even if using nonhydrolyzable analogs of IP 3 or GTPγS), the resultant Ca2+
signal shows poor correlation with that found at fertilization (Miyazaki et al. 1990 ;
Swann and Ozil 1994 ; Galione et al. 1994 ). Altogether, these data suggest that eggs
contain the signaling machinery that is normally associated with cell surface recep-
tors, but do not necessarily mean that the fertilizing sperm actually use these signal-
ing cascades to trigger embryo development.
Xenopus eggs have been shown to activate in response to external application of
sperm components (Iwao et al. 1994 ), and peptides containing an RGD sequence
(an integrin-binding site found in a sperm-associated protein) cause a Ca2+ increase
(Iwao and Fujimura 1996 ). In addition, Xenopus sperm contain a protein, xMDC16,
that is a member of the metalloprotease-/disintegrin-/cysteine-rich protein family
(Shilling et al. 1997 ). Peptides containing a sequence of the disintegrin domain of
xMDC16 trigger a Ca2+ increase and activation when applied near the egg surface
(Shilling et al. 1998 ). Such peptides are able to bind and stimulate potential integrin-
like egg surface receptors (Foltz and Shilling 1993 ). These results suggest that in
Xenopus the fertilization Ca2+ signal is in fact generated via surface membrane
interactions between the gametes (Iwao 2000 ). However, up to now no receptor has
been identified for either the RGD-containing sequence or the xMDC16. Incubation
in the presence of a soluble RGD peptide also induces Ca2+ increase in bovine eggs
(Campbell et al. 2000 ), but again, the receptors that have been identified on mam-
malian eggs so far seem to be involved in gamete binding and fusion, rather than the
stimulation of Ca2+ release (Wassarman et al. 2005 ).
The sperm content hypothesis claims that the fertilization Ca2+ signal is triggered
by a substance in the sperm that diffuses into the ooplasm following sperm-egg fusion
(Fig. 1.3). In mouse eggs, gamete fusion precedes the Ca2+ oscillations by 1–3 min,
which is consistent with the idea that a factor diffuses into the ooplasm and induces
Ca2+ release (Lawrence et al. 1997 ). Microinjection of a crude extract from the sperm
head into mammalian eggs was found to stimulate a series of low- frequency Ca2+
oscillations identical to those seen at fertilization (Swann 1990 ; Wu et al. 1997 ;
Machaty et al. 2000 ). Initially, it was suggested that the extract’s active factor might
be IP 3 (Tosti et al. 1993 ); later, in a number of invertebrate species, nitric oxide (NO;
Kuo et al. 2000 ) and nicotinic acid adenine dinucleotide phosphate (NAADP; Lim
et al. 2001 ) were also proposed. Heat or trypsin treatment of the mammalian sperm
extract abolished its Ca2+-inducing activity suggesting that the mammalian sperm fac-
tor was a protein, and adding the extract to the eggs did not cause Ca2+ release either
indicating that the factor was specific to the sperm cytosol (Swann 1990 ). The activity
of the sperm factor is a general phenomenon in mammalian species (Swann et al.
1998 ), and furthermore, extracts isolated from frog or chicken sperm cause Ca2+ oscil-
lations in mouse eggs (Dong et al. 2000 ). The success of intracytoplasmic sperm
injection in mammals, where membrane interaction between the gametes is bypassed
by the direct injection of the sperm into the ooplasm, also supports the notion that it is
a factor in the sperm head that stimulates Ca2+ changes at fertilization.
1 Egg Activation at Fertilization