Could modifications of signalling pathways activated after ICSI induce a potential risk of epigenetic defects?

Abstract

A calcium signal during oocyte or egg activation is a conserved event in virtually all species analyzed so far. This signal, that is in the form of calcium oscillations in mammals, is spatially and temporally controlled and is mainly supported by calcium release from internal calcium stores, but how it is triggered after fertilization is far from understood. The sperm factor hypothesis of egg activation postulates that sperm delivers a calcium-releasing factor into the egg following sperm-egg fusion. Among the many potential sperm factors, PLCzeta is the strongest bona fide sperm factor candidate. However, how sperm-oocyte fusion occurs prior to PLCzeta delivery and oocyte activation is not entirely known. We propose in the first part of this review the possibility that other pathways such as those involving G-proteins, tyrosine kinases or integrins could be activated besides sperm factor injection and could be upstream mechanisms involved in later embryonic development. Among different assisted reproductive technologies (ARTs), intracytoplasmic sperm injection (ICSI) is considered as the best and easiest therapeutic technique to circumvent severe male infertility. Although most reports are reassuring, some recent data suggest a greater incidence of abnormalities in children conceived by ART compared with those conceived normally. Spatio-temporal signals may be missing or abnormal during ICSI, perhaps because membrane fusion and signalling events are bypassed. We discuss in the second part of this review the hypothesis that potential perturbations during the ICSI procedure may have repercussions on epigenetic processes, inducing not only alterations of embryonic development, but also diseases in young children and, perhaps, in adults.

Full text

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Could modifications of signalling pathways activated after
ICSI induce a potential risk of epigenetic defects?
BRIGITTE CIAPA*,1 and CHRISTOPHE ARNOULT2
1UMR CNRS 8195 Centre de Neurosciences Paris-Sud (CNPS), Université Paris XI, Orsay Cedex and
2Inserm U.836, Université Joseph Fourier, Grenoble, France
ABSTRACT A calcium signal during oocyte or egg activation is a conserved event in virtually all
species analyzed so far. This signal, that is in the form of calcium oscillations in mammals, is
spatially and temporally controlled and is mainly supported by calcium release from internal
calcium stores, but how it is triggered after fertilization is far from understood. The sperm factor
hypothesis of egg activation postulates that sperm delivers a calcium-releasing factor into the egg
following sperm-egg fusion. Among the many potential sperm factors, PLCzeta is the strongest
bona fide sperm factor candidate. However, how sperm-oocyte fusion occurs prior to PLCzeta
delivery and oocyte activation is not entirely known. We propose in the first part of this review the
possibility that other pathways such as those involving G-proteins, tyrosine kinases or integrins
could be activated besides sperm factor injection and could be upstream mechanisms involved in
later embryonic development. Among different assisted reproductive technologies (ARTs), intra-
cytoplasmic sperm injection (ICSI) is considered as the best and easiest therapeutic technique to
circumvent severe male infertility. Although most reports are reassuring, some recent data
suggest a greater incidence of abnormalities in children conceived by ART compared with those
conceived normally. Spatio-temporal signals may be missing or abnormal during ICSI, perhaps
because membrane fusion and signalling events are bypassed. We discuss in the second part of
this review the hypothesis that potential perturbations during the ICSI procedure may have
repercussions on epigenetic processes, inducing not only alterations of embryonic development,
but also diseases in young children and, perhaps, in adults.
KEY WORDS: fertilization, oocyte, egg, ICSI, calcium, epigenetics
Activation of the mammalian egg after fertilization
Many reviews have been published to describe how adhesion
and fusion of sperm and egg at fertilization induces the formation
of a zygote that develops into a new individual. In mammals, the
oocyte that can be fertilized is arrested at metaphase of second
meiotic division after ovulation. The metabolism of this oocyte is
low, and if no fertilization occurs the oocyte will die. This arrest of
the cell cycle is due to high activities of MAPK ("M-phase activat-
ing protein kinase") and MPF ("mitosis promoting factor"), the
latter being a complex between cdc2 kinase and cyclin B. After
fertilization, oocyte activation triggers inactivation of these two
kinases, exit of meiosis, and start of embryonic development
(Ducibella and Fissore, 2008). Normally, egg activation after
fertilization starts from the point of sperm-egg interaction and
Int. J. Dev. Biol. 55: 143-152 (2011)
doi: 10.1387/ijdb.103122bc
THE INTERNATIONAL JOURNAL OF
DEVELOPMENTAL
BIOLOGY
www.intjdevbiol.com
*Address correspondence to: Brigitte Ciapa. 8195 Centre de Neurosciences Paris-Sud (CNPS), GDR 2688, Université Paris XI, Bât 445, F-91405 Orsay Cedex,
Paris, France. e-mail: brigitte.ciapa@u-psud.fr
Accepted: 26 October 2010. Final author corrected PDF published online: 8 June 2011.
ISSN: Online 1696-3547, Print 0214-6282
© 2011 UBC Press
Printed in Spain
Abbreviations used in this paper: ART, assisted reproductive technologies; Ca,
calcium; ICSI, intracytoplasmic sperm injection; MAPK, M-phase activating
protein kinase; MPF, mitosis promoting factor.
fusion. In all species studied so far, from invertebrate to mam-
mals, the process of egg activation has not yet been clearly
identified, but several hypotheses have been proposed in the
literature and include: 1) interaction between oocyte receptor(s)
and sperm ligand(s); 2) formation of a pore between the two
gametes allowing the diffusion into the oocyte of calcium and/or
one (or several) spermatic factor(s); 3) insertion in the oocyte
plasma membrane of spermatic plasma membrane elements
such as calcium channels (Fig. 1). These different hypotheses are
not exclusive and several, if not all, could be employed at

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144 B. Ciapa and C. Arnoult
the origin and the cause of any chromosomal anomaly
detected in children conceived by ICSI. Contrary to
classical in vitro fertilization, interactions between
membranes of sperm and oocyte that normally occur
after fertilization are bypassed in ICSI. We discuss in
this review the possibility that events triggered dur-
ing or after fertilization and normally induced at the
plasma membrane level could be absent or abnor-
B
A
fertilization.
In developed countries, it is estimated that 1–3% of children
are conceived by ARTs (Assisted Reproductive Technologies), in
order to treat human infertility. Several factors associated with
assisted reproductive methods might increase the malformation
rate, which is regularly discussed in the literature. Firstly, chromo-
somal anomalies may be carried by the gametes used, which
could also be at the origin of male or female infertility itself.
Secondly, methods used to stimulate fertility and leading for
example to superovulation or in vitro maturation of gametes have
clearly been reported to be able to perturb epigenetics and
genomic imprinting, particularly in the extra-embryonic tissues
(Laprise, 2009). ART techniques include intra-uterine insemina-
tion, in vitro fertilization (IVF) and ICSI (intracytoplasmic sperm
injection). ICSI is considered today as the best and easiest
therapeutically technique to circumvent severe male infertility.
Injection of a sperm into an egg circumvents natural sperm
selection and could indeed transfer any chromosomal anomaly
from paternal origin. Finally, ICSI could also introduce foreign
material such as culture medium or exogenous DNA and infec-
tious material (Chan et al., 2000). It is therefore difficult to know
mally generated during ICSI and could have an impact on late
development and even after birth.
The fertilization calcium signal
In mammals and all other species analyzed so far, fertilization
always triggers in the egg a rapid and transient increase in
intracellular free calcium (Cai), and various recent reviews have
been published that describe and discuss the nature and the role
of this ionic signal in egg activation and development of the
embryo. In mammals, this “calcium signal” is in the form of Cai
oscillations, the frequency and intensity of which vary with the
species (Swann and Yu, 2008; Ducibella and Fissore, 2008).
They are generally observed in human, but seem to be altered
after conventional ART or after ICSI as discussed below.
In general, calcium acts as a second messenger through
multiple pathways and controls a wide range of cellular functions,
from exocytosis to gene expression and even cell apoptosis
(Scharenberg et al., 2007; Berridge, 2009) Cai signals are inter-
preted by specific proteins that are responsible for the onset of
development. Many of these proteins are protein kinases (CaMKII,
Fig.1. Interaction between sperm and egg after fertili-
zation that is bypassed after ICSI. (A) Three possibilities
that are not exhaustive. (1) Interaction between sperm
ligands and receptor(s) expressed at the surface of the
oocyte. Molecules of the sperm plasma membrane (spPM)
can bind receptors of the plasma membrane of the oocyte
(ovPM): fertilin, cyritestin (ADAM3), elements of the extra-
cellular matrix (EM) such as fibronectin, these elements
being ligands of integrins, protein complement regulator
the receptor of which are unknown, or diverse other
molecules (X) which could bind receptor with tyrosine
kinase activity (R-tyrK) or receptors bound to G proteins (R-
Gpt). Tetraspanins CD9 and CD91 could interact with
integrins, R-tyrK or R-Gpt as it is the case in T lymphocytes.
All these molecules can be part of a complex capable of
inducing adhesion and fusion between gametes, but also
enzymatic activities (Enz Act) such as tyrosine kinase that
can be involved or not in triggering a Ca signal. (2) Role of
Ca channels. Calcium channels are already present in the
plasma membrane of the oocyte. Moreover, several classes
of calcium channels are also present in the sperm plasma
membrane and are inserted into the oocyte plasma mem-
brane after sperm fusion. The sperm channels may be
activated after their incorporation. Both oocyte and sperm
channels would trigger Ca increase in the vicinity of ER
large enough to modulate the shape of the Cai signal. (3)
Formation of a pore between gametes allowing the diffu-
sion of one (or several) sperm factor(s). (B) Ca signal and enzymatic activities regulate epigenetic modifications at early times after fertilization.
Enzymatic activities and Ca signal normally induced after sperm-egg interaction would constitute two outputs (output 1 and 2 respectively) of well-
defined duration and amplitude. This would regulate epigenetic modifications that also need to occur in a precise window of time and amplitude for
normal development. Altered outputs 1 and 2 triggered after ICSI would lead to abnormal epigenetic modifications.

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ICSI and activating pathways 145
PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly
regulated by Cai and varies not only during oocyte maturation but
also at early times following fertilization (Ducibella and Fissore,
2008). In order to avoid activation of unwanted cellular process,
Cai increases are spatially and temporally controlled and a wide
range of calcium channels are present in the plasma and reticu-
lum membranes to achieve their spatial and temporal control
(Boulware and Marchant, 2008; Rizzuto et al., 2009).
It has become a dogma that this event is necessary and
sufficient to activate eggs in all species that have been studied so
far, since blocking the calcium increase with Ca2+ buffers inhibits
egg activation and inducing an artificial calcium increase acti-
vates the egg. It is important to determine the criteria that are used
to judge egg activation. For example, in almost all schema
proposed in the literature to explain how a sperm activates an egg,
the calcium signal is upstream of all events that lead to develop-
ment of the zygote. However, only "early events" are chosen as
the criteria for judging egg activation, i.e. those normally occurring
before blastocyst formation. Whether the Cai signalling events
have an impact on long-term embryonic development has not
been well examined. Furthermore, Ca independent signalling
pathways could be activated concomitantly with the Cai signal,
and be involved in events occurring after blastocyst formation.
Several questions are therefore apparent: 1) could abnormal
development or diseases be induced after ICSI because of an
altered Ca signal transduction? 2) Could signal transduction
pathways that are bypassed by ICSI none-the-less have a role to
play during development?
Calcium release from the endoplasmic reticulum
Increase in Cai in the egg is mostly due to a release of Cai from
endoplasmic reticulum (ER). Several types of Ca channels are
involved: inositol trisphosphate receptors (IP3-R) (Berridge, 2009),
ryanodine receptors (RyR) whose activation is regulated either by
Ca2+ influx or by cADPr (cyclic adenosine diphosphate ribose)
(Yin et al., 2008), or channels activated by NAADP (nicotinic acid
adenine dinucleotide phosphate) (Patel et al., 2010). In hamster,
only IP3-R seems to be involved (Miyazaki et al., 1992). In mouse,
RyR, although present and functional in oocytes, would not be
solicited during oocyte activation, and only IP3-R, aggregated in
clusters of the RE, would be necessary (Fitz Harris et al., 2003).
NAADP could play a key-role in Cai signal origin in various species
but has not yet been demonstrated to be involved in mammals
(Santella et al., 2004). In human oocytes, propagation of the Cai
signal differs from that observed in other mammals, whatever the
protocol of fertilization. This signal is characterized by a rapid
increase in Cai at the periphery of the oocyte, followed by a slower
increase in Cai deeper in the cytoplasm. The fact that ryanodine-
sensitive calcium stores are located at the periphery of the mature
oocyte suggests a role at the origin of the fertilization calcium
signal in human (Sousa et al., 1996). In any case, the IP3-R1
seems to be required to generate Cai oscillations and activate
mammalian eggs (Ducibella and Fissore; 2008), and how IP3
production is activated after fertilization is then an important issue
that needs to be clarified.
Role of calcium channels located in the plasma membrane
The oocyte is an excitable cell, and hyperpolarisation occurs
during fertilization in mammals (Tosti, 2010). Dependence be-
tween calcium modification and plasma membrane potential has
been argued by Miyazaki (1989) suggesting a role of voltage-
dependent channels in the Ca rise. Voltage dependent calcium
channels are present in plasma membrane: L type Ca channels
have been described in bovine oocytes and T-type channels in
early mouse embryos (Tosti, 2010). Ca channels could be acti-
vated during interaction with the sperm, as described in sea urchin
(David et al., 1988), which would allow a sufficient amount of
calcium into the oocyte to stimulate the surrounding IP3-Rs and
generate the Ca signal. These Ca channels could also be directly
coupled to IP3-R or RyR as it is the case in muscle (Berridge,
2009). Insertion of sperm Ca channels into the oocyte plasma
membrane during sperm / egg fusion has also been proposed
several years ago in sea urchin (McCulloh and Chambers, 1992)
but this hypothesis has never been proven, even in this species.
These activated channels may behave as described above.
Mammalian sperm contain various Ca channels: VOC ("voltage
activated channels"), ROC ("receptor-activated channel") and
SOC ("store-activated-channel"),that are involved during the
acrosome reaction (Tosti, 2010). Finally, a TRP-3 type Ca chan-
nel has recently been involved in the fertilization process in C
elegans. Mutations of this channel lead to sterility of hermaphro-
dite and male animals, sperm being motile but incapable of
fertilizing oocytes (Xu and Sternberg, 2003). It is important to note
that Ca channels of this type are expressed in human (Castellano
et al., 2003). Whether some types of Ca channels of the oocyte
plasma membrane are involved or not in the initiation of the
fertilization Cai signal, they play an important role in “refilling”
intracellular calcium stores to maintain them full during several
hours and later in the fertilized mammalian oocyte (Ducibella and
Fissore, 2008).
Role of mitochondria
Mitochondria are present in large number in eggs and act as a
relay in Cai signalling at fertilization. They are necessary to
maintain Cai oscillations for a long time in mouse oocytes
(Dumollard et al., 2009). Studies on ascidians and mouse have
shown that sperm-triggered Cai oscillations are transduced into
mitochondrial Cai signals that stimulate mitochondrial respiration.
Mitochondrial Cai uptake can substantially buffer cytosolic Cai
concentration and the concerted action of heterogeneously dis-
tributed mitochondria in the mature egg may modulate the spa-
tiotemporal pattern of sperm-triggered Cai oscillations (Dumollard
et al., 2009). Furthermore, inadequate redistribution of mitochon-
dria at MII, unsuccessful mitochondrial differentiation, or de-
creased mitochondrial transcription induce significantly lower
rate of embryo development (Dumollard et al., 2009), even after
activation by intracytoplasmic sperm injection (Nagai et al., 2006).
Alterations of mitochondrial calcium signalling mechanisms could
then not only affect the fertilization Cai signal and, consequently,
embryonic development, but also lead the oocyte toward apopto-
sis, a cellular death program that involves mitochondria and is
used by oocytes when they are not fertilized (Dumollard et al.,
2009).
The sperm factor
The sperm factor hypothesis has strongly been built up by the
fact that human oocytes can start embryonic development after
ICSI, since during this procedure, sperm / egg interaction is short-

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146 B. Ciapa and C. Arnoult
circuited. This hypothesis also comes from the fact that sperm
extracts obtained in invertebrate such as ascidia as well as in
mammals such as human are capable of generating Ca oscilla-
tions in mouse, hamster, human or sea urchin oocytes, and even
in somatic cells. These sperm extracts are then non species
specific, which suggests that the sperm factor is universal and
identical in all species. The sperm factor would be either soluble,
or insoluble and associated with the perinuclear material, and is
thermo labile since it loses its activating effect after heating
(Swann and Yu, 2008). During more than fifteen years, several
candidates have been proposed, some have then been clearly
forgotten, others remaining to be proved to be "the good one". As
an example, a 33 kDa proteic factor was purified, then cloned and
called “oscillin”. Oscillin was described as specifically localized in
the cytosol, in the sperm equatorial plate, the zone that is involved
during gametes interaction. Testicular slices from sterile men
correlated with the absence of oscillin protein using anti-oscillin
antibodies. It was also shown that oscillin could bind an oocyte
component, creating a complex capable of activating both RyR
and IP3-R. In fact, oscillin was a glucosamine-6-phosphate deami-
nase present in various other tissues and was then clearly ruled
as being the "sperm factor" (Ducibella and Fissore, 2008).
A truncated form of c-kit (tr-kit), a tyrosine kinase receptor of
the PDFG receptor family, has also been proposed as a potential
sperm factor, since it could trigger oocyte activation, with cell
cycle resumption, MAPK kinase inhibition, extrusion of the sec-
ond polar body and cortical granule exocytosis. This 24 kDa
protein was proposed to bind another tyrosine kinase fyn, which
would lead to PLC stimulation (Sette et al., 2002). However,
several points must be clarified to verify this hypothesis. One can
for example ask why tr-kit is expressed in the mid-piece of the
flagella and not in the equatorial zone which fuses first with the
oocyte It has also never been shown whether tr-kit injection
induces the same Cai oscillations as those induced at fertilization.
However, it is possible that tr-kit injection induces activation of
pathways involving tyrosine kinase of the src or fyn family, which
can bind integrins such as 61 (Streuli and Akhtar, 2009),
molecules that are expressed in the oocyte as described below.
As mentioned above, the IP3-Rs are necessary to generate Cai
oscillations. The IP3 that activates these receptors comes from
hydrolysis of phoshatidylinositol bisphosphate (PIP2) by a phos-
pholipase C (PLC). PLCs form a family of molecules that respond
to defined signalling pathways. For example, PLC is down-
stream G-protein receptors while PLC belongs to a tyrosine
kinase signalling pathway (Berridge, 2009).The identity of the
isoform of the particular PLC upstream the IP3-R could reveal
information concerning the cellular pathway that is stimulated at
fertilization. Various data led to the hypothesis that sperm would
directly inject a PLC into the oocyte following sperm-oocyte fusion
which would produce IP3 in the oocyte. A PLC was purified and
cloned in 2002 from a mouse testis EST library: PLC. PLC has
since been cloned in monkey and human suggesting the ubiquity
of PLC in Mammals. This PLC does not contain any particular
domain usually found in other PLCs, PLC or PLC, and such as
SH2, SH3 or PH. PLC is the smallest PLC known to date.
Expression of mRNA encoding this protein in mouse oocytes
induces Cai oscillations similar to those observed after fertilization
in this species. In human, injection of complementary RNA of
human PLC in oocytes that were not activated by ART or ICSI
induces blastocyst formation. Cai oscillations induced by PLC
exclusively appear in M phase, after its nuclear localization.PLC
shows such a very high sensitivity to Ca that it should be active
even at rest, but the binding of its C2 domain to particular
phosphatidylinositides (PI3P et PI5P) would regulate its activity.
All these results strongly argue that PLC is the long sought after
sperm factor (Swann et al., 2006; Swann and Yu, 2008). How-
ever, it is intriguing that PLC is not expressed in animals where
the genome has been sequenced such as sea urchin, ascidian,
Drosophila, and C-elegans. Therefore, PLC cannot be the uni-
versal sperm factor and perhaps only exits in vertebrates. An
unfertile knock out male mouse would be the best way to confirm
that PLC is the sperm factor but so far this has not been
performed successfully.
Calcium signalling after ICSI
ICSI seems to produce a pattern of Cai oscillations slightly
different from those described after normal fertilization. In human,
the first Cai oscillations triggered after ICSI are truncated and
delayed, in comparison with those observed after SUZI ("sub
zonal insemination"), which however does not alter the following
Cai oscillations (Tesarik and Sousa, 1994). In mouse, the spatial
distribution of the Cai rise (Nakano et al., 1997) and duration of Cai
oscillations (Kurosawa and Fissore, 2003) are not equivalent in
eggs subjected to ICSI and those subjected to IVF. Therefore,
alterations of the calcium signal can be observed at very early
times (alterations of first peaks) or in the number and frequency
of following oscillations.
Alterations of early Ca peaks
In ICSI, the sperm is immobilized and then taken by aspiration
in the injection pipette. The injecting pipette is pushed against the
zona, permitting its penetration and thrusting forward to the inner
surface of the oolemma. As the point of the pipette reaches the
approximate center of the egg, a break occurs in the membrane.
This is reflected by a proximal flow of cytoplasmic organelles and
the spermatozoon back up into the pipette. The sperm is then
slowly ejected back into the cytoplasm, with oocyte cytoplasm.
Rupture of the oocyte plasma membrane is a sine qua non
condition of fertilization success. In mouse, a first Cai rise is
inevitably produced by calcium entering through the opening of
the plasma membrane during the ICSI procedure and affects the
duration, intensity and spatial heterogeneity of the subsequent
first Cai responses, depending on the intensity of this first non
physiological peak (Miyazaki, 1999). ICSI then immediately in-
duces a Ca influx, which can also be obtained after injection of
medium that does not contain any sperm, but which would not
activate the oocyte by itself (Tesarik and Sousa, 1994; Tesarik,
1998). This Ca influx induced after ICSI seems necessary but not
sufficient to activate the oocyte. The different methods used to
immobilize the sperm during ICSI (pipeting, squeezing, piezo
application) seem to influence the beginning of the Cai oscilla-
tions. All these techniques damage the sperm membrane. The
piezo method is the one that induces Ca oscillations more quickly,
and induces the highest percentage of fertilization success
(Yanagida et al., 2001). Rupture of the sperm membrane is likely
required to release one or several factor(s) involved in generating
the Cai oscillations (Marangos et al., 2003). These results suggest

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ICSI and activating pathways 147
that the ICSI procedure itself takes the place of the "start signal"
that is normally activated after sperm and egg interaction.
Could this difference have any incidence on later develop-
ment? The impact could be direct, by affecting Ca-dependent
mechanisms such as those depending on Ca-calmodulin kinases
which themselves will transduce an altered signal. ICSI entails
breaching the plasma membrane, which results in a Cai influx
while plasma membrane integrity is being restored. The entering
Cai can trigger spontaneous Ca2+ transients, and such high
frequency Cai oscillations have been observed after fertilization
when membrane integrity is disrupted (Ozil, unpublished obser-
vations cited in Ozil et al., 2006), which could well have an impact
on late embryonic development, as discussed below. Finally, a
few births obtained after ICSI in oocytes that were previously
activated by Ca ionophore have recently been reported. This
procedure was used when repeated fertilization failed after "clas-
sical" ICSI (Eldar-Geva et al., 2003; Chi et al., 2004; Heindryckx
et al., 2008; Taylor et al., 2010). It is important to note that this was
done directly in human without any previous result obtained in
mouse or any other animal species used in laboratories. Ca
ionophore triggers a huge increase in Cai due to Ca influx from the
external medium and leaks from intracellular stores. The pattern
of Cai oscillations obtained in these conditions is unknown, but is
likely abnormal.
Abnormal Cai signals could easily be due to the fact that, during
ICSI, the sperm factor is being delivered from a different place and
at a different rate than during physiological fertilization (as dis-
cussed by Nakano et al., 1997). ICSI could modify the early
fertilization Ca peaks by altering Ca channels that trigger these
peaks. For example, increase in the Cai level during ICSI proce-
dure as described previously could modify the activity of IP3-R
which are sensitive to calcium (Berridge, 2009). Finally, Ca
channels of the oocyte plasma membrane described above could
be bypassed after ICSI.
Modifications in the number and period of Cai oscillations
Cai oscillations induce MPF inactivation by acting on a Ca/
calmodulin upstream MPF and on mechanisms involved in cyclin
B destruction. Alterations in the fertilization Cai oscillations could
then alter mechanisms that depend on MPF activity and that are
responsible for early embryonic development (Ducibella and
Fissore, 2008). The level of MPF varies with oocyte maturation
and can modify the pattern of Cai oscillations. However, this
impact would be indirect in mammals, as shown in mouse, where
MPF determines the timing of formation of the pronucleus which
itself regulates Cai oscillations after fertilization (Ducibella and
Fissore, 2008). The number of Cai oscillations also controls early
morphological events such as cortical exocytosis and recruitment
of maternal mRNA (Ducibella and Fissore, 2008), and acts on cell
composition in blastocysts by altering the trophectoderm cell
number in mouse (Bos-Mikich et al., 1997). Finally, modifications
of Cai level can also alter protein synthesis in mouse oocyte (Bos-
Mikich et al., 1995).
The pattern of Cai oscillations depends on the maturity of each
gamete used during ICSI. Several results have shown that the
ROSI ("round spermatid injection") or ELSI (elongated spermatid
injection) techniques can lead to oocyte activation in mouse,
hamster and human that could occur without normal Cai oscilla-
tions (Yazawa et al., 2000). However, another report shows that
the ROSI technique applied to 58 couples did not give any birth
(Urman et al., 2002). ICSI applied on oocytes that were matured
in vitro can also lead to births (Liu et al., 2003), but to our
knowledge, no result has been obtained in human concerning the
impact of oocyte maturation on the Cai signal.
Alterations of late development might well occur when Cai
oscillations are not accurately generated (Ozil et al., 2006). A
precocious interruption of natural regime of Cai oscillations alters
the incidence of implantation whereas hyper-stimulation of Cai
signalling events compromises post-implantation development
inducing a greater variability in the weight of such induced
offspring, which indicates a reduced developmental competence
of the blastocysts. More importantly, analysis of global patterns of
gene expression by microarray analysis revealed that approxi-
mately 20% of the transcripts were mis-regulated when too few
oscillations were generated in the embryo. Expression Analysis
Systematic Explorer (EASE) analysis indicated that genes prefer-
entially involved in RNA processing and polymerase II transcrip-
tion were differentially affected, and those involved in cell adhe-
sion mis-expressed which could explain reduced implantation.
3% of the transcripts were mis-regulated following hyper-stimula-
tion, and EASE analysis indicated that genes preferentially in-
volved in metabolism were differentially affected. All these results
strongly suggest that altering the fertilization Cai signal can have
long-term effects on both gene expression and development to
term (Ozil et al., 2006).The frequency of the Cai oscillations
seems to be modulated by the sperm content in a dose-dependent
manner, which suggests that the concentration of some sperm
factor within the sperm is a crucial element of normal fertilization
and oocyte activation (Faure et al., 1999). Sperm used for ICSI
and containing low amounts of the sperm factor PLC can trigger
enough Cai oscillations sufficient to initiate development but
insufficient to support development to term (Ozil et al., 2006).
Globozoospermia is a severe pathology characterized by the
absence of the acrosome and thus sperm are unable to cross the
zona pellucida. Although ICSI bypasses zona pellucida crossing,
oocyte activation does not occur with these deficient sperm, and
this lack of activation seems to be correlated with PLC absence.
Recent reports suggest that reduced amounts or abnormal forms
of PLC could be overcome by activating the sperm-injected
oocytes with Ca ionophore (Kyono et al., 2008, Tejera et al., 2008,
Taylor et al., 2010). Although this treatment could lead to high
rates of fertilization and an ongoing pregnancy, we may wonder
what the risks are from both a probable abnormal Ca signal as
already discussed above and a highly abnormal sperm material
since a high percentage of sperm DNA fragmentation is associ-
ated with this pathology.
Signalling cascades upstream or independent of the
fertilization calcium signal
If the fertilization Ca signal is necessary and sufficient to trigger
egg activation, it is therefore essential to know how it is triggered
in the egg from the starting point of sperm/egg interaction, and we
have mentioned above the different hypotheses that have been
proposed in the literature. The first point to consider is that any
single cascade could not only lead to a calcium signal but could
also contain elements capable of activating Ca-independent
pathways. The second point is that sperm could indeed straightfor-

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148 B. Ciapa and C. Arnoult
wardly induce a calcium signal, for example through the injection
of a sperm factor, but this does not exclude necessarily the
stimulation in parallel of other pathways, independent of the Ca
signal. The possibility that events may be generated at the plasma
membrane level besides this Ca signal after sperm - egg interac-
tion and may have an impact on late development has never been
envisaged. If that were the case, it would question the role of
various signalling elements such as G proteins that have generally
been tested by looking only at their ability to induce a Ca signal, and
not on their capability to act on other events that do not necessarily
depend on calcium. Finally, since the interaction between mem-
branes of sperm and oocyte that normally occurs after fertilization
is bypassed after ICSI, putative signalling pathways generated
from the plasma membrane would also be bypassed, or abnor-
mally stimulated. Since ICSI leads to apparently normal develop-
ment, one must admit that all events necessary to trigger develop-
ment have been stimulated by ICSI. Organization into nanodomains
and clusters of different signal transduction circuits, for example in
lipid rafts that have been suggested to play a crucial role in egg
activation (Sato et al., 2006), allows fine regulation of duration and
intensity of various in vivo responses, including Ca or MAP kinase
signals (Harding and Hancock, 2008). Therefore, all putative
signalling pathways proposed below generated by integrins,
tetraspanins, GPI or R-G proteins may have been stimulated after
ICSI by crosstalks and/or feedback loops.
Role of tyrosine kinase or G-protein receptors
This hypothesis comes from the fact that several molecules
expressed at the sperm surface could bind to the oocyte plasma
membrane and behave as ligands of oocyte receptors. Various G
proteins (Gs, Gi 1-3, Go) are expressed in the mouse oocyte.
Moreover, microinjection of GTPS, a GTP analogue, leads to
oocyte activation while microinjection of GDPS, which inhibits G-
proteins, cancels the Cai signal in hamster. These results are in
favor of a G-protein receptor in these species (Runft et al., 2002),
but neither receptor of this type nor putative ligand has ever been
identified. However, G-proteins could be indirectly activated through
other pathways implying tyrosine kinase activity. Similarly, the
presence and role of a tyrosine-kinase receptor has never been
demonstrated (Runft et al., 2002).
Role of the Gq family G proteins in mouse eggs activation has
been investigated by looking at their ability to initiate calcium
release, cortical granule exocytosis, recruitment of maternal mR-
NAs, and cell cycle resumption, but whether they can affect later
development is not known. Similarly, injection of SH2 domains or
addition of tyrosine kinase inhibitors to block the normal stimulation
of PLC, or src like kinases also fails to stop sperm induced Cai
oscillations in mouse eggs (Mehlmann and Jaffe, 2005). These
results only strongly suggest that neither PLC not src like kinase
are sufficient to transduce a Cai signal, but cannot exclude the idea
that they may be involved in later development, or need to be
associated with another event to generate the fertilization Ca
signal. As a matter of fact, although Src-family PTKs are not
required for fertilization-induced calcium oscillations in mouse,
activation of these kinases in distinct regions and at specific times
does play a critical role in development of the zygote (Luo et al.,
2009). PTKs and G-protein receptors are linked to MAPK activation
(Shaul and Seger, 2007), which itself may act on Cai oscillations
(Matson and Ducibella 2007).
Integrins, tetraspanins and glycosylphosphatidylinositol- pro-
teins
In mammals, several sperm proteins that could behave as
specific ligands of oocyte receptors have been identified. Ligands
of integrins, that are elements of the extracellular matrix such as
fibronectin or fertilin, have largely been described in the literature
to be expressed at the sperm surface.Fertilin is composed of two
transmembranal subunits,  and . The  subunit contains a
disintegrin domain that could bind an integrin of the egg surface,
while the  subunit presents in its intracellular domain a helical
motif that is also found in viral fusion proteins, which would allow
the membranes of the two gametes to fuse. Fertilin has been
cloned in various mammals including human. This molecule be-
longs to a family of 39 membrane proteins that have been named
ADAMs (“A Disintegrin And Metalloprotease domain”). Some of
them are expressed in the sperm, and the best studied are fertiline
 (ADAM2) and cyritestine (ADAM3). Mutagenesis of these pro-
teins leads to infertile male mice, which suggests a role of these
proteins in the process of sperm-egg interaction. However, other
ADAMs could also be involved. A new sperm protein, named
Izumo, has been identified as required for sperm fusion with
oocyte. Although its oocyte receptor is unknown, this receptor
could play a role in egg activation (Rubinstein et al., 2006). The
search of the putative receptor of fertilin has led to many articles
and controversies over the last years. It has been known for many
years that various integrins, glycoproteins that are are constituted
of two subunits bound non-covalently,  and , are expressed at
the oocyte surface and that inhibition of these molecules with
antibodies or specific ligands can alter fertilization. However,
knockout experiments have shown that all female mice that do not
express 1 integrins, including 2, 3, 5, 6, 9 and v subunits,
or that do not express the v3 or v5 integrins, are fertile.
Moreover, combination of these KO with inhibition of the function
of other integrins does not alter fusion between the two gametes.
All these results argue against a role of integrins in egg activation
(Rubinstein et al., 2006). Recently, the integrin 9 subunit has
been suggested to play a role in sperm-egg binding and fusion
(Vjugina et al., 2009). Finally, it has been reported that the
tripeptide RGD, that binds some integrins, can trigger an increase
in Cai and even Cai oscillations in the bovine oocyte (Campbell et
al., 2000).
CD molecules are cell surface molecules that are associated
with the immune system and could also be involved in the fertiliza-
tion process Expression of complement regulatory proteins such
as DAF ("Decay activating factor") or CD55 have been described
on oocytes, but no role in activation has ever been attributed to
these molecules. The tetraspanin CD9 plays a role at fertilization
since Cd9 -/- female have a severe reduced fertility. Another
tetraspanin, CD81, is also weakly expressed in the oocyte, but its
role in adhesion and/or fusion has not yet been clarified, although
fertility of female mice lacking CD81 is reduced (Fabryova and
Simon, 2009; Rubinstein et al., 2006).
The role of integrins at fertilization is not easy to decipher,
considering the redundancy of these proteins that often function
inside multi-protein complexes and in association with other types
of receptors including tyrosine kinase receptors and G-protein
receptors (Streuli and Akhtar, 2009). Integrins can mediate, inside
micro-domains, a few interactions with other proteins including
CD9 and CD81. As an example, CD81 is associated in T lympho-

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ICSI and activating pathways 149
cytes with 41 in the mobility functions of these cells (Kolesnikova
et al., 2004). Palmitoylation of CD9 and CD81, as well as that 3,
6, and 4 integrin subunits favors their association inside protein
complexes (Yang et al., 2004). As a matter of fact, the tetraspanin
CD9 would be involved in human and mouse gamete fusion by
helping the formation of clusters containing the integrin 61
(Fabryova and Simon, 2009). Tetraspanins can interact with G-
protein receptors (Little et al., 2004). Integrins can also alter cellular
behaviour through the recruitment and activation of signalling
proteins such as non-receptor tyrosine kinases including focal
adhesion kinase (FAK) and c-Src that form a dual kinases complex.
The FAK-Src complex can phosphorylate various adaptor proteins
such as p130Cas and paxillin. Multiple integrin-regulated linkages
exist to activate FAK or Src and to interact with other adaptor
molecules such as ILK (integrin-linked kinase), PINCH (particularly
interesting new cysteine-histidine rich protein) and Nck2 (a ty-
rosine kinase adaptor protein). Integrin signalling tightly and coop-
eratively interacts with receptor tyrosine kinase signalling path-
ways via all these molecules (Streuli and Akhtar, 2009).
Finally, female mice that do not express Pig-A, an enzyme
necessary to the binding of glycosylphosphatidylinositol- proteins
(GPI), are infertile and produce oocytes that cannot fuse with
sperm, which suggests a role of GPI during gamete fusion (Alfieri
et al., 2003).
In conclusion, all these proteins that can act as part of multicom-
ponent complexes would be capable not only of inducing adhesion
and fusion of gametes, but also to start various types of signalling
pathways including a Cai signal, tyrosine kinase or MAP kinase
activities. It is clear that abolishing one of them only, for example
after KO experiment, would not necessarily lead to inhibition of egg
activation, if another protein inside this complex has a similar role,
as it is the case for integrins, or if one missing leg would not refrain
the centipede to walk.
Fertilization and epigenetic modifications
Nature and function of epigenetic mechanisms
Gene expression is not only regulated by genetic mechanisms
but also by epigenetic modifications. The epigenetic information is
not encoded by the DNA sequence itself but by reversible modifi-
cations of DNA and/or associated histones, and can be transmitted
from cell to daughter cell and even from one generation to the next
generation. DNA methylation and histone modifications interplay
to control gene expression (Vaissière et al., 2008). Enzymes that
induce histone modifications include acetyltransferases (HATs),
arginine and lysine methyltransferases (HMTs), ubiquitin ligases
and peptidyl arginine deaminase (PAD) (Nottke et al., 2009). DNA
methylation is a process that allows the transfer of a methyl group
from S-adenosylmethionine to cytosine residues in the CpG di-
nucleotides by different methyltransferases (Cheng et al., 2008).
The entire genome is subjected to epigenetic modifications during
gametogenesis and embryogenesis. Particularly, some of these
processes are activated very rapidly after sperm-egg fusion and
could well depend on the signalling pathways described above.
DNA demethylation after fertilization
DNA methylation is totally erased in germinal cells and then re-
established during gametogenesis (Feil, 2009). Each cell has two
copies of each gene, one of maternal origin and the other of
paternal origin. However, there are genes for which one copy only
is expressed, the other being repressed. This genomic repression
depends on the parental origin, and is called the genomic imprint-
ing. Before fertilization, one of the alleles, maternal or paternal, is
silenced, and then transmitted to the lineage in this conformation.
In human, this process is essential for placenta and embryo
development, but mechanisms at the origin of this process are still
not well understood (Koerner and Barlow, 2010). In mammals,
fertilization triggers an active and rapid DNA demethylation of the
paternal genome, followed by a passive DNA demethylation of the
maternal genome. The entire genome is then methylated again
during embryonic development (Feil, 2009). The occurrence and
the extent of DNA demethylation of the paternal genome is
however controversial among different mammalian species
(Abdalah et al., 2009). The mechanisms at the origin of the active
demethylation of the paternal genome, and those protecting the
maternal genome at the very beginning of embryogenesis, are
just beginning to be deciphered, but still poorly understood.
PGC7/Stella, that was initially identified as a gene predomi-
nantly expressed in primordial germ cells (PGCs) has been
shown to protect the maternal genome from demethylation
(Nakamura et al., 2007). Very recently, the component of the
elongator complex5 Elp3 (also called KAT9) has been reported to
be involved in this process although these authors do not have
evidence indicating that Elp3 directly acts upon DNA as a DNA
demethylase. Similar results were obtained with the elongator
components Elp1 and Elp4. Interestingly, the role of the SAM
radical domain in Elp3 seems to be crucial (Okada et al., 2010).
This domain is present in the radical SAM superfamily proteins
that use S-adenosylmethionine (SAM) to catalyse a variety of
radical reactions. SAM is substrate for an activating enzyme, the
pyruvate formate-lyase (PFL) activase, PFL leading to the pro-
duction of acetyl-CoA + formate from pyruvate + CoA (Wang and
Frey, 2007). Interestingly, selection between glucose and lactate
by a regulated compartmentation of pyruvate in neurons and
astrocytes depends on cytosolic NAD+/NADH redox conditions
(Cerdan et al., 2006), which are themselves crucial for mouse
early development (Dumollard et al., 2009). Mitochondria are
deeply involved in the regulation of the redox state of cells and of
eggs in particular and as mentioned above, also modulate Cai
oscillations in fertilized eggs (Dumollard et al., 2009). This may
look like a “drawer hypothesis”, but the fertilization Cai signal,
mitochondria and redox potential might act together to modify
epigenetic information during early development since DNA pa-
ternal demethylation occurs at early times after fertilization (Santos
et al., 2005).
Histone modifications after fertilization
Although the female haploid genome is associated with his-
tones in a somatic-like chromatin structure in the fertilizable
oocyte, the spermatic chromatin is highly condensed and tran-
scriptionally inert before fertilization but becomes transcription-
ally competent in the male pronucleus (Miller et al., 2010).
Modifications of DNA-binding histones that form the nucleosome,
H2A, H2B, H3 and H4, occur after fertilization and seem to differ
between maternal and paternal DNA (Wu et al., 2008). More
importantly, these modifications could occur very rapidly after
fertilization. For example, histone H4 is highly acetylated in the
paternal genome at an early stage and remains acetylated at a low

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150 B. Ciapa and C. Arnoult
level in the maternal pronucleus in early one-cell embryos (Wu et
al., 2008). A very quick loss of histone H2A variants H2AL1 and
H2Al2 from the paternal pericentric heterochromatin regions
occurs after sperm-egg fusion (Wu et al., 2008).
Could an abnormal signal generated after fusion of sperm and
egg have an impact on early epigenetic modifications? In mam-
mals, the formation of the pronucleus (PN) is under the control of
MAPK activity and perhaps depends on the frequency of Cai
oscillations (discussed in Dulcibella and Fissore, 2008). It is not
known why this event occurs 3-4 hours after expulsion of the
second polar body in mammalian zygotes, but this delay could be
the time required to reconfigure the sperm chromatin (Morgan et
al., 2005). Therefore, an altered timing in the formation of PN may
result in altered reprogramming of the male genome. Modifica-
tions in Cai signalling might also alter activities of enzymes
involved in histone epigenetic modifications since they are turned
on rapidly after fertilization (Santos et al., 2005) and as described
above. The activity of the PAD enzymes is calcium dependent
(Holbert and Marmorstein, 2005). For example, PADI4 is a Cai -
dependent peptidylarginine deiminase enzyme that can function
to antagonize arginine methylation levels by demethylimination
(Hagiwara et al., 2005). Histone deacetylase also interacts with
Cai /calcineurin -mediated signalling pathways during T cell
activation and proliferation (Im and Rao, 2004) and cardiac gene
expression (Sanna et al., 2005). Moreover, stimulation of the
integrin pathway could lead to histone epigenetic modifications
after fertilization. For example, integrin engagement increases
histone H3 acetylation and reduces histone H1 association with
DNA in murine lung endothelial cells, thus inducing global sensi-
tivity of DNA to nuclease digestion, which reflects alterations in
chromatin structure (Rose et al., 2005).
ICSI and epigenetic defects
Can the risk of epigenetic defects increase after ICSI? Most of
the studies give reassuring data meanwhile recommending a
more thorough investigation because the level of defects is
nevertheless statically higher in babies born through ART and
among the different reasons, imprinting defects may be prepon-
derant (Palermo et al., 2009). Heterogeneity of the cohorts used
in all these studies renders difficult quantification of such risks
linked to ART and particularly to ICSI, and it seems difficult to
appreciate whether risks are due to the ART procedures, infertility
and/or age of parents. More particularly, ART has been associ-
ated with an increase in the frequency of apparition of the Prader-
Willi or d’Angelman syndrome, that are due to defects of the
genetic imprinting of one region of the chromosome 15, and of the
Beckwith Wiedemann syndrome, that is due to epigenetic abnor-
malities in a cluster of genes that are submitted to imprinting in
one region of the chromosome 11 and triggers a macrosomy and
an increased risk of neoplasy (Manipalviram et al., 2009). Per-
centages reported in these studies remain however very low and
the number of reported cases insufficient for deducing any con-
clusion.
Defects in methylation reprogramming of PG3 and APC genes
(Zechner et al., 2009) and of the methylation status of H19
Imprinting Control region (ICR) and H19 gene expression (Fauque
et al., 2007) have been reported to occur during ART in human.
Very few data have been reported where epigenetic modifications
after ICSI vs normal or ART fertilization have been studied. The
pattern of histone methylation seems to differ between ART and
ICSI in mouse (Van der Heijden et al., 2009) and even in human
(Qiao et al., 2010). In rats, the demethylation dynamics of the
paternal genome at pronuclear-stage is impaired when ART are
used (Yoshiwara et al., 2010). Sperm chromatin remodeling after
ICSI is more asynchronous than in ART in mouse embryos (Ajduk
et al., 2006).
Conclusion
Signalling pathways involved during ICSI may be different in
duration and intensity from those stimulated after normal fertiliza-
tion. Could this have a repercussion on epigenetic processes?
Are epigenetic modifications of the genome linked to Cai signal-
ling and/or to pathways independent of Ca generated at fertiliza-
tion and could they be altered after ICSI because they did not
occur in the natural frame of time or at a right intensity? Could
modifications of the initial Cai signal, for example after activation
of the oocyte with Ca ionophore, have any incidence on epige-
netic mechanisms? And finally, is it possible that epigenetic
defects induced at the time of fertilization lead to diseases and
cancers that would occur only at late adult age?
Epigenetic defects can lead to major pathologies including
various syndromes linked to chromosome instabilities or mental
retardation (Zhao et al., 2007). More specifically, alterations of
parental imprinting have been associated with cancer (Esteller,
2007). Moreover, it is accepted that epigenetic alterations that
lead to some forms of cancers are probably linked to environmen-
tal factors and ageing (Feinberg, 2007). ICSI has been performed
since 1992. Children born that way at that time have then just
reached the age of young adults. A large number of cancers and
of neuronal and muscular diseases are revealed only in adults. Is
it possible that children born after ICSI or ART have epigenetic
alterations that are undetectable during young life but develop
some kind of cancer by ageing or after exposure to some environ-
mental factors?
All reports on the frequency of abnormalities in children con-
ceived by ART look currently reassuring. However, we believe
that more studies should be performed to study molecular mecha-
nisms regulating epigenetic, and more particularly focusing on
potential relationships with calcium signalling. Studies on epige-
netic modifications after fertilization and putative alterations in-
duced by ART should be encouraged. Finally, this review points
out the fact that mechanisms of egg activation still remain unclear
and need further investigation.
Acknowledgements
B Ciapa is supported by CNRS and C Arnoult is supported by CNRS
(centre de la recherche scientifique) and ANR (Agence nationale de la
recherche). We thank Robert Feil for his critical comments and Alex
McDougall for amending our manuscript.
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ICSI and activating pathways 153
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5 yr ISI Impact Factor (2009) = 3.253