Oocyte activation after intracytoplasmic injection of
mature and immature sperm cells
Jan Tesarik
Laboratoire d'Eylau, 55 Rue Saint-Didier, 75116 Paris, France
The inactivation of metaphase-promoting factor
(MPF), leading to reactivation of the oocyte cell
cycle after fertilization, is one of the most
important results of oocyte activation in the
case of intracytoplasmic injection of mature or
immature sperm cells. Oocyte activation is a
cell signalling event that is likely to imply
receptors at the oocyte plasma membrane and
a signal transduction pathway involving calcium
and protein phosphorylation/dephosphorylation. The typical calcium signal during oocyte
activation in mammals takes the form of calcium
oscillations. Intracytoplasmic sperm injection
(ICSI) is associated with a slightly different
pattern of calcium oscillations in comparison
with normal fertilization. Available data suggest
that sperm cytosolic factor(s) play a role of
oscillator, modulating the properties of the
oocyte's intracellular calcium stores, whereas
the role of trigger, normally realized by spermoocyte interactions at the level of their respective
cell surfaces, can be supplemented in the conditions of ICSI by an artificial calcium influx
generated by the procedure itself. Delayed onset
and an abnormal form of the oocyte activationpromoting calcium signal are the two known
molecular abnormalities of human oocyte
activation; they can lead to fertilization failure
and are also suspected to be at the origin of
various embryo abnormalities. The impact of
oocyte activation abnormalities on future development increases when oocytes are fertilized
with immature sperm cells (spermatids) because
the chromatin of these cells is less protected
than sperm chromatin against a rapid action of
the oocyte's MPF. If oocyte stimulation by the
Human Reproduction Volume 13 Supplement 1 1998
sperm calcium oscillation-promoting activity,
which first appears at the round spermatid stage
of human spermatogenesis, is insufficient to
cause a rapid inactivation of MPF, premature
condensation of spermatid chromatids may lead
to aneuploidy.
Key words: fertilization/intracytoplasmic sperm
injection/oocyte activation/spermatid/spermatocyte
Introduction
Following the first reported human pregnancies
and births obtained after fertilization of oocytes
by intracytoplasmic sperm injection (ICSI; Palermo
et al, 1992) and the conclusive demonstration of
the unusually high efficiency of ICSI in terms of
fertilization and pregnancy rates (Van Steirteghem
et al, 1993), this method has been increasingly
used in the treatment of infertility, including cases
in which spermatozoa have to be recovered from
the epididymis or the testis and thus have not
completed the process of epididymal maturation
(Silber, 1994; Devroey et al, 1996). Pregnancies
and births have also been obtained in mice after
transfer of embryos developing from oocytes electrofused or injected with round spermatids (Ogura
et al, 1994; Kimura and Yanagimachi, 1995a) or
secondary spermatocytes (Kimura and Yanagimachi, 1995b) and in humans after transfer of embryos
developing from oocytes injected with elongated
(Fishel et al, 1995a, 1996) and round (Tesarik
et al, 1995a, 1996) spermatids (reviewed in
Tesarik, 1996a).
The problem of oocyte activation is of paramount
importance to all these techniques of micromanipu-
© European Society for Human Reproduction & Embryology
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J.Tesarik
lation-assisted reproduction. In fact, the failure of
oocyte activation appears to be the main cause of
fertilization failure after ICSI (Sousa and Tesarik,
1994). Moreover, the events occurring during
oocyte activation are believed to condition embryo
quality for many subsequent cell cycles (Swann
and Ozil, 1994), and irregularities of these events,
that may occur after ICSI, are suspected to be
the cause of different embryonic abnormalities,
including chromosomal ones (Tesarik, 1995). This
paper outlines the current understanding of the
cellular and molecular mechanisms involved in
oocyte activation and focuses on those elements
that are likely to be affected by factors associated
with the intra-ooplasmic injection technique and
with the use of immature sperm cells for fertilization.
Mechanism of oocyte activation
The mature human oocyte has its cell cycle blocked
at the metaphase of the second meiotic division
(metaphase II). By analogy with other mammalian
species, the metaphase II block of human oocytes
is supposed to be maintained by persisting high
activities of metaphase-promoting factor (MPF).
From the functional point of view, this factor is
the same as that controlling the progression of the
cell cycle in somatic cells, in which its activity
changes periodically to drive cell nuclei to metaphase during each mitotic division. Consequently,
any nucleus introduced into a metaphase II oocyte
will be driven to metaphase unless MPF has been
inactivated. This MPF inactivation is one of the
principal events of oocyte activation which
unblocks the cell cycle of the future zygote, allows
the female nucleus to complete the second meiotic
division and protects the male nucleus from
entering metaphase prematurely in response to
the oocyte's MPF. The triggering of the cortical
reaction is another consequence of oocyte activation which, under normal conditions when the zona
pellucida is not severed, is important in setting in
motion a polyspermy-preventing mechanism. The
fact that progression of preimplantation embryonic
development can be modified by artificially manipulating the conditions of oocyte activation (Swann
and Ozil, 1994) suggests that, in addition to MPF
inactivation and the cortical reaction, many other
118
cellular functions are influenced by events occurring during oocyte activation.
There is no doubt that physiological oocyte
activation is induced by the spermatozoon at fertilization. However, oocytes can also be activated by
a number of artificial physical and chemical stimuli
unrelated to spermatozoa (parthenogenetic activation). Of course, oocyte activation produced by
such unnatural stimuli will often be abnormal or
incomplete. Whether or not such phenomena
should still be referred to as oocyte activation will
thus depend on the outcome measures followed in
each experimental study.
The mechanism whereby the spermatozoon
induces oocyte activation can be studied at different
levels. One of the most important questions relating
to this mechanism is how the spermatozoon generates the immediate triggering stimulus which starts
the whole cascade of reactions ultimately leading to
oocyte activation. How this introductory stimulus is
subsequently transduced via intracellular signalling
pathways towards the corresponding effectors is
another fundamental question. Finally, it is crucial
to know how these signalling pathways operate in
space and time to ensure an ordered stimulation
of different targets within the oocyte.
Triggering stimulus
Experimental studies performed in various animal
species suggest that the spermatozoon triggers
oocyte activation by acting at a receptor or several
receptors on the oocyte plasma membrane; these
receptors appear to be coupled to a G-protein or
to a protein tyrosine kinase (Jaffe, 1990; Miyazaki
et al, 1990; Ciapa and Epel, 1991; Foltz et al,
1993; Moore et al, 1993; Abassi and Foltz, 1994;
Shilling et al, 1994; Iwao and Fujimura, 1996).
A sperm receptor has been characterized in sea
urchin eggs as a transmembrane glycoprotein sharing some sequence similarity with a heat-shock
protein, hsp70 (Foltz et al, 1993). A protein
capable of triggering electrical responses in eggs
similar to those occurring at fertilization was
isolated from Urechis sperm acrosomes (Gould
and Stephano, 1987). In mammals, no candidate
protein for this function has as yet been identified.
However, the plasma membrane of mammalian
oocytes contains the adhesion molecules integrins
Immature sperm and oocyte activation
(Tarone et al, 1993), whereas the posterior head
region of mammalian spermatozoa (the region by
means of which the first tight contact between the
sperm and oocyte plasma membranes is established
during fertilization) bears an integral plasma membrane glycoprotein termed fertilin (Primakoff et al.,
1987; Blobel et al, 1992) whose (3-subunit contains
an integrin-binding domain (Wolfsberg et al., 1993;
Myles et al, 1994). Moreover, human spermoocyte binding has been shown to be inhibited by
immunobeads coated with peptides containing the
RGD (Arg-Gly-Asp) sequence known to serve as
ligands for integrins (Fusi et al., 1992). Since
integrins mediate transmembrane signal transduction in many cell types (Yamada and Miyamoto,
1995), sometimes involving a mobilization of intracellular calcium (Shankar et al, 1993) which also
is a dominant feature of oocyte activation (see
below), integrins present on the oocyte surface
may be stimulated by sperm fertilin and are thus
good candidates for oocyte activation-triggering
receptors. Oocyte activation accompanied by
intracellular calcium release could also be induced
by RGD-containing peptides in Xenopus (Iwao and
Fujimura, 1996).
The recent development of ICSI, and especially
the finding that calcium responses similar to those
observed during normal fertilization (Taylor et al,
1993) can be obtained after ICSI (Tesarik et al,
1994), has often been interpreted as arguing against
the surface-receptor hypothesis of oocyte activation
triggering and in favour of the so-called solublesperm-factor hypothesis. In fact, it was shown as
early as the mid-1980s that sea urchin oocytes can
be activated by direct intracytoplasmic injection
of a soluble sperm extract (Dale et al, 1985),
without any previous contact between the gamete
surfaces. The possibility of activating oocytes by
injecting soluble sperm extracts has subsequently
been confirmed in various mammalian species
(Swann, 1990, 1994) including the human (Homa
and Swann, 1994; Dozortsev et al, 1995a). A
highly conserved protein capable of carrying out
this action in different mammalian species has
been characterized (Parrington et al, 1996). The
existence in spermatozoa of a soluble oocyteactivating factor explains why essentially normal
oocyte activation events can be achieved after ICSI
although the eventual function of oocyte surface
receptors is obviously by-passed. However, the
current experience with ICSI does not imply that
such receptors play no role in human oocyte
activation occurring in situations other than ICSI.
On the contrary, the observations that some oocytes
fail to activate after ICSI although the injected
spermatozoon has completely disintegrated and
thus released all its cytoplasmic contents into the
ooplasm (Sousa and Tesarik, 1994) and that such
oocytes can still be activated by artificially increasing their calcium load with an ionophore (Tesarik
and Sousa, 1995a), suggest that the oocyte activation process after ICSI is unusually vulnerable and
may not follow exactly the same pattern as the
physiological oocyte activation involving the gamete surface interaction. The lack of surface receptor
participation in the oocyte activation events after
ICSI can also provide an explanation for the
observation that the calcium responses recorded
during oocyte activation after ICSI are slightly
different from those observed when oocytes are
fertilized by subzonal insemination, which does not
bypass the gamete surface contact and interaction
(Tesarik and Sousa, 1994). However, the reason
for this difference may be related to different rates
and locations of release of the sperm soluble factor
in the two situations. Taken together, the data on
human oocyte activation that are available, suggest
that the fertilizing spermatozoon activates the
oocyte both by acting at a receptor on the oocyte
surface and by releasing a soluble factor into the
ooplasm, although the latter can eventually assume
the totality of the task, provided that it is aided by
artificial stimuli brought about, intentionally or
unintentionally, by the ICSI procedure itself (see
below).
Signal transduction pathways
In all animal species studied so far, the oocyteactivating signal uses calcium as a second messenger. In mammalian oocytes, the source of calcium
participating in these signalling events is predominantly intracellular, although calcium influx from
the extracellular space is also involved (Swann
and Ozil, 1994). Releasable calcium is stored in
cells within calcium stores (presumably mainly
endoplasmic reticulum) within which it is accumu119
J.Tesarik
lated against a concentration gradient through an
energy-consuming action of a calcium pump and
from which it can be released along the existing
concentration gradient without an energy requirement by opening calcium channels in the calcium
store membrane. Two types of calcium-store calcium channels are known: one of them is a receptor
for inositol 1,4,5-trisphosphate (IP3) and the other
is a receptor for ryanodine. In addition to the plant
alkaloid ryanodine, the ryanodine receptor/calcium
channel also opens in response to a physiologically
occurring ligand, cyclic ADP-ribose. Both types
of channels can open as a result of an increase in
the calcium concentration outside the stores in their
immediate vicinity; this phenomenon is known
as calcium-induced calcium release (CICR). The
emptying of a calcium store usually generates a
signal that is conveyed to the plasma membrane,
leading to the opening of its own calcium channels
and resulting in calcium influx; this phenomenon
is known as capacitative calcium entry.
The current data about the type and function of
calcium stores in mammalian oocytes are somewhat contradictory. There is little doubt that IP3sensitive stores are present in oocytes (Miyazaki
et al, 1993; Kline and Kline, 1994; Fissore et al,
1995; Miyazaki, 1995). On the other hand, ryanodine-sensitive stores have been detected by some
workers (Swann, 1992; Ayabe et al, 1995; Yue
et al, 1995; Sousa et al, 1996a) but not by others
(Miyazaki et al, 1993; Kline and Kline, 1994;
Fissore et al, 1995). The presence of ryanodinesensitive calcium stores in oocytes and their role
in the oocyte activation mechanism have been
recently demonstrated in humans (Sousa et al,
1996a), and these calcium stores have been suggested to participate, together with IP3-sensitive
stores, in a two-store mechanism of calcium oscillations (Tesarik et al, 1995b; Tesarik and Sousa,
1996).
How the fertilizing spermatozoon induces the
release of calcium from oocyte intracellular stores
is still a matter of debate. It is possible that the
spermatozoon acts directly on only one type of
calcium store within the oocyte, so that the resulting
calcium discharge activates CICR from the other
type of store. Data obtained with hamster and
mouse oocytes suggest that the sperm action at the
120
oocyte surface leads to the activation of a Gprotein (Miyazaki, 1988; Moore et al, 1993).
It can be postulated that the G-protein-mediated
activation of phosphoinositide-specific phospholipase C leads to hydrolysis of plasma membrane
phosphoinositides thus yielding IP3 which, in its
turn, will release calcium from the IP3-sensitive
stores followed by CICR from the ryanodinesensitive stores. In agreement with this hypothesis,
ryanodine-insensitive, but thimerosal- and presumably IP3-sensitive, calcium stores have been
detected in the cortical region of human oocytes
(Sousa etal, 1996a). Diacylglycerol, another product of the phosphoinositide breakdown, may then
activate protein kinase C (PKC), which is supposed
to be involved in the down-regulation of the IP3mediated calcium release mechanism, and thus
facilitates the subsequent return of the intracellular
free calcium concentration ([Ca2+];) to the basal
level (Swann et al, 1989; Miyazaki et al, 1990).
This sequence of events may underlie the sharp
transient [Ca 2+ ]; increase referred to as a calcium
spike. Notwithstanding, other mechanisms involving oocyte plasma membrane receptors and their
ligands, sperm soluble factors, IP3-sensitive calcium stores and ryanodine-sensitive calcium stores
also may come into play. The observations that
the injection of sperm extracts into oocytes can
induce a rapid [Ca2+]j increase (Swann, 1990,
1994) do not mean that soluble sperm factors are
the only physiological oocyte-activation triggers.
In fact, it appears difficult to determine an exact
dosage of the sperm factors injected in the form
of sperm soluble extracts. Consequently, there is
still some doubt as to whether the dose of sperm
cytosolic factors required for the rapid response
observed after the injection of sperm extracts can
actually be delivered by a single spermatozoon at
fertilization. Moreover, the microinjection technique used for the deposition of sperm extracts
into oocytes may itself modify the oocyte response
to the injected factors. These uncertainties do not
question the role of such factors in oocyte activation
but warn against simplifications that lay too much
emphasis on only one aspect of the oocyte-activation mechanism and ignore its probable complexity.
Relatively little is known about how the calcium
signal is transduced to downstream elements of
Immature sperm and oocyte activation
Metaphase arrest
MPF
(cyclin/cdc2)
Metaphase arrest exit
CSF
(c- mos product)
Sperm
CAM kinase II
Calcium
PKC
Figure 1. Schematic representation of the probable mechanism by which sperm-induced oocyte activation drives the oocyte to
exit from metaphase II arrest. The metaphase II arrest is maintained by persisting high activities of metaphase-promoting factor
(MPF; consisting of the regulatory component termed cyclin and the cyclin-dependent kinase cdc2) and cytostatic factor (CSF)
that is a product of the c-mos proto-oncogene. The calcium signal generated by the fertilizing spermatozoon activates protein
kinases, of which calmodulin-dependent protein kinase II (CAM kinase II) and protein kinase C (PKC) have been suggested as
the most likely candidates. The kinase activation is then postulated to lead to phosphorylation of MPF and CSF and their
subsequent inactivation (for a more detailed review, see Swann and Ozil, 1994).
the cell activation cascade. Changes in protein
phosphorylation, involving calmodulin-dependent
protein kinase II (Lorca et al, 1993) and PKC
(Bement and Capco, 1991; Grandin and Charbonneau, 1991), are probably implicated (Figure 1).
The phosphorylation both of MPF and of a cytostatic factor (CSF) that is involved in the maintenance of MPF activity may play a role in the
mechanism of inactivation of MPF (Watanabe
et al, 1991; Lorca et al, 1993).
Spatiotemporal characteristics
Unlike fish, echinoderms and frogs, in which the
fertilizing spermatozoon induces a single transient
[Ca2+]j increase, the oocyte-activation signal in
mammals and some other species consists of a
series of repetitive short calcium spikes known as
calcium oscillations. In human oocytes, the sperminduced calcium oscillations can last for up to 5 6 h (Taylor et al, 1993; Sousa et al, 1996b); the
frequency with which individual calcium spikes
appear ranges between one spike every 2 min to
one every 35 min and is highly variable from
oocyte to oocyte but relatively stable in a single
oocyte during the whole calcium oscillation period
(Taylor et al, 1993; Tesarik and Sousa, 1994;
Tesarik et al, 1995b; Sousa et al, 1996b). However, in most mammalian species studied so far
(reviewed in Swann and Ozil, 1994) including the
human (Tesarik and Sousa, 1994), the very early
[Ca2+]j reaction of oocytes to the fertilizing spermatozoon shows a unique pattern that is slightly
different from the bulk of the calcium oscillation
period; in human oocytes, this early calcium reaction is characterized by an initial group of three to
six rapidly occurring calcium spikes which is then
followed by lower-frequency spikes for the rest of
the calcium oscillation period (Tesarik and Sousa,
1994). The sperm-induced calcium oscillations of
mammalian oocytes are believed to bear a unique,
frequency-encoded signal that is necessary for the
complete oocyte-activation process (Vitullo and
Ozil, 1992; Ozil and Swann, 1995). In agreement
with this hypothesis, incomplete activation with a
failure of polar body extrusion was observed after
artificial induction of a single [Ca 2+ ], increase in
oocytes of the protostome worm Cerebratulus
lacteus that, similarly to mammalian oocytes, also
require calcium oscillations for complete activation
(Strieker, 1996).
In view of the long duration of the sperminduced calcium oscillations in human oocytes, it
121
J.Tesarik
is clear that the fertilizing spermatozoon must
deliver to the oocyte not only the immediate trigger
to induce the oscillations but also a message
modifying the properties of one or several of the
main elements involved in the regulation of calcium
homeostasis (calcium pumps and channels in the
plasma and calcium store membranes) to facilitate
that kind of calcium exchanges between individual
compartments that are required for the maintenance
of calcium oscillations. We may use the term
'oscillator' for this latter message. The consideration of the trigger and oscillator as two distinct
physiological entities is based on the observations
that human ICSI can sometimes lead to the development of a single monotonous sperm-dependent
[Ca2+]j increase (Tesarik et ah, 1994) or bring
oocytes into a condition in which they are ready
to develop calcium oscillations but do not do so
unless stimulated with an additional, ionophoreinduced [Ca2+]j increase (Tesarik and Testart,
1994). The former situation can be explained
by an isolated action of the trigger without the
oscillator, whereas the latter results from the action
of the oscillator without the natural trigger. Sperm
cytosolic factors, among which oscillin (Parrington
et ah, 1996) is the only one identified as yet, are
the most probable candidates for the oscillator
function.
Experimental data obtained with human oocytes
(Sousa et ah, 1996a) suggest that calcium oscillations are maintained by periodic release and resorption of calcium from IP3-sensitive stores, localized
predominantly in the oocyte cortex and subcortex,
and ryanodine-sensitive stores localized mainly in
the central ooplasm (Tesarik and Sousa, 1996),
thus conforming to a previously formulated twostore calcium oscillation model (Tesarik et ah,
1995b). An alternative two-store model for calcium
oscillations in mammalian oocytes does not involve
the ryanodine-sensitive calcium stores but assumes
the existence of two functionally distinct types of
IP3-sensitive calcium store, one responsible for
calcium entry and the other giving the regenerative
spike (Berridge, 1996). According to the former
model, the ability of the oocyte to maintain calcium
oscillations depends on the differential sensitivity
of the two types of calcium stores to CICR, such
that an initial limited calcium release from the
122
cortical stores (presumably sensitive to IP3) acts as
a detonator for CICR from the central, ryanodinesensitive stores from which most of the calcium
released during each calcium spike originates.
Because the cortical stores have a higher sensitivity
to CICR than the central ones, the zone of increased
[Ca2+]j propagates from the initial focus in the
cortex around the oocyte periphery, with the likely
participation of the capacitative calcium entry,
before extending to the central ooplasm where, on
the other hand, [Ca2+]j remains elevated for the
longest time at the end of each calcium spike
(Tesarik et ah, 1995b). The latter model (Berridge,
1996) can be expected to work in a similar way
but the ryanodine-sensitive calcium stores are substituted by a unique type of IP3-sensitive calcium
store. The sensitivity to CICR of one or both of
the oocyte's calcium stores may be modified by
the sperm-derived oscillator, as suggested by the
unique pattern of the spatial propagation of sperminduced calcium waves compared to those induced
by artificial stimuli such as thimerosal or ryanodine
(Tesarik et ah, 1995b; Sousa et ah, 1996a).
Phosphorylation by PKC has been suggested to be
part of the mechanism controlling the characteristics of the ryanodine-sensitive calcium stores in
human oocytes because a sustained stimulation of
PKC inhibits sperm-induced calcium oscillations
and prevents any calcium response of oocytes to
ryanodine, but not to thimerosal, whereas inhibition
of PKC interferes with the return of [Ca 2+ ]; to
basal values during the sperm- and ryanodineinduced calcium oscillations, but not during the
thimerosal-induced calcium oscillations (Sousa
et ah, 1996c).
Particular questions relating to oocyte
activation after intracytoplasmic injection of
mature and immature sperm cells
Skipping gamete surface interaction: does it
make a difference?
The experience with ICSI indicates that injected
spermatozoa can activate oocytes completely and
induce calcium oscillations similar to those seen
during normal fertilization. Using the preceding
vocabulary, we can say that, in the absence of
sperm-derived triggers that probably require an
Immature sperm and oocyte activation
interaction between the gamete surfaces to act,
the sperm-derived oscillators can be sufficient to
support oocyte activation provided this process is
triggered by an independent stimulus. It is very
likely that the calcium influx produced by the ICSI
procedure itself can serve as such an auxiliary
stimulus (Tesarik and Sousa, 1995b), although it
is insufficient to trigger oocyte activation in the
absence of spermatozoa (Tesarik et al, 1994).
The amount of calcium entering the oocyte is
proportional to the degree of ooplasmic aspiration
during the ICSI procedure (Tesarik and Sousa,
1995b). The degree of ooplasmic aspiration is
difficult to quantify objectively, and even the degree
of calcium influx without intentional ooplasmic
aspiration is likely to vary from laboratory to
laboratory as a result of differences in the quality
of the injection needle and in the technical aspects
of the oocyte plasma membrane disruption. This
explains why fertilization results were related to
the degree of aspiration in some (Palermo et al,
1995; Tesarik and Sousa, 1995b) but not all
(Mansour et al, 1996) laboratories. Oocyte activation after ICSI also works better when spermatozoa
are mechanically damaged by the microinjection
needle just before ICSI (Dozortsev et al, 1995b;
Fishel et al, 1995b; Van den Bergh et al, 1995)
to facilitate sperm disintegration in the ooplasm
and the release of oocyte-activating factors.
In addition to the truncation of the typical initial
sperm-induced calcium signal, which is common
with ICSI (see above), two types of more severe
oocyte-activation
abnormalities have been
reported: an unusual delay in the beginning of the
sperm-induced calcium oscillations and substitution of calcium oscillations with a long nonoscillatory [Ca2+]j increase (Tesarik et al, 1994;
Tesarik and Testart, 1994). By analogy with other
animal species, such abnormalities may cause serious developmental consequences. For instance,
failure of the second polar body extrusion, leading
to the formation of tripronucleate zygotes after
ICSI, may result from an abnormal calcium signal,
similar to that described in protostome worms
(Eckberg and Miller, 1995; Strieker, 1996). Moreover, the delayed onset of the oocyte-activation
signal is likely to lead to a delayed or incomplete
inactivation of MPF (Tesarik, 1996b) which, in its
turn, can cause abnormalities of the second meiotic
anaphase leading to chromosomal abnormalities
in the future embryo (Tesarik, 1995). Diverse
cytological abnormalities, involving sperm aster
formation, pronuclear development, spindle formation and cell division, were observed in rhesus
monkey oocytes fertilized by ICSI and were suggested to be at least partly due to abnormalities of
calcium signalling (Hewitson et al, 1996). Even
though all of these relationships remain hypothetical in the human, they deserve attention, particularly in view of the recently published data about
the incidence of chromosomal abnormalities after
ICSI (In't Veld et al, 1995).
Use of immature sperm cells: the significance
of calcium oscillations revisited
The generally accepted view that the oscillatory
character of the sperm-induced calcium signal is
required for the optimal efficiency of the fertilization process in mammals, in terms of the developmental potential of the resulting embryo, has been
challenged by recent reports of relatively elevated
birth rates after fertilization of mouse oocytes
with isolated nuclei of round spermatids and secondary spermatocytes (Kimura and Yanagimachi,
1995a,b). These findings mean that either calcium
oscillations can be induced in oocytes by factors
associated with spermatid and spermatocyte nuclei,
or the oscillatory character of the sperm-induced
calcium signal is not important for normal oocyte
activation, at least in the mouse. The preliminary
data with regard to spermatid conception in humans
do not raise a similar dilemma. In fact, the three
childbirths reported so far after spermatid conception in humans (Tesarik et al, 1995a, 1996; Fishel
etal, 1995a, 1996) were all obtained after injection
of whole spermatids into oocytes, not just their
isolated nuclei. Moreover, the injection of human
spermatids, but not primary and secondary spermatocytes, into human oocytes sensitizes the calcium
response mechanism of the oocytes in a way very
similar to the injection of mature spermatozoa
(Sousa et al, 1996d). It should also be noted that
a limited amount of ooplasm accompanied the
spermatid and spermatocyte nuclei injected into
the oocytes in the above mouse experiments, and
this might be enough to sensitize the oocytes for
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J.Tesarik
the development of calcium oscillations. To answer
these questions, an analysis of [Ca 2+ ], following
injection of spermatid and spermatocyte nuclei into
mouse and rabbit oocytes is needed.
Testicular spermatozoa may also be slightly
different from ejaculated spermatozoa with regard
to the activity or releasability of oocyte-activating
factors because the former appear to require a
more aggressive mechanical disruption before ICSI
than the latter to achieve optimal fertilization
results (Palermo et al, 1996).
Table I. Consequences of inadequate timing of oocyte
activation after injection of mature and immature sperm cells
Type of sperm cell
injected
Beginning of activation
Too early
Too late
Spermatocyte II
Spermatid
Triploidy
Pronuclear
asynchrony
Ageing
PCC, aneuploidy
Spermatozoon
Pronuclear
asynchrony
Ageing
PCC = premature chromosomal condensation.
Co-ordination of the effects of oocyte activation
on the male and the female nucleus: a newly
emerging problem
One of the reported abnormalities of oocyte activation following ICSI is its delayed onset (Tesarik
et al, 1994), although such a delay has not always
been observed (Nagy et al., 1994). Consequently,
the male nucleus can be exposed for some time to
MPF that is not inactivated until the oocyte is
activated (Figure 1). A short exposure of male
gamete nuclei to active MPF is necessary even for
normal fertilization with mature spermatozoa, since
Borsuk (1991) has shown that the sperm nuclear
membrane breakdown in the ooplasm is a MPFdependent event, whereas the subsequent pronuclear development is MPF-independent. This observation underscores the importance of a fine coordination between the exposure of the male nucleus to oocyte cytoplasmic factors and the progression of oocyte activation. Unlike mature
spermatozoa, whose chromatin is highly condensed
and stabilized by specific nuclear proteins, the
protamines, so that the oocyte MPF is unable to
drive sperm nuclei to metaphase prematurely, in
developing spermatids the process of histone/protamine exchange is still ongoing. Spermatid nuclei
are thus much more sensitive to untimely oocyte
activation events than nuclei of mature spermatozoa (Tesarik, 1996b). If MPF is allowed to drive
spermatid nuclei to metaphase, the consequences
for future embryo development are disastrous
because the prematurely condensed chromatids
bypass the S-phase, associate with microtubules
and can be extruded from the oocyte in an uncontrolled manner as soon as the metaphase II block is
overcome by subsequent oocyte activation (Kimura
124
and Yanagimachi, 1995a). This situation leads
inevitably to the development of aneuploidy
(Table I). Exactly the same mechanism was used
with success for chromosomal reduction of secondary spermatocytes injected into mouse metaphase
II oocytes, resulting in the development of diploid
zygotes (Kimura and Yanagimachi, 1995b).
On the other hand, a premature onset of oocyte
activation, while the male nucleus is still absent
or not fully accessible to ooplasmic factors, can
cause situations in which the male nucleus receives
a truncated developmental message as compared
to the female nucleus. This might result in a
developmental imbalance between the two nuclei
(Table I). It remains to be determined whether
such an imbalance is the cause of the nuclear
abnormalities, such as the persistence of an unusually small male pronucleus (Ogura et al, 1994;
Tesarik and Mendoza, 1996) or the prolonged
nuclear syngamy (Tesarik and Mendoza, 1996)
observed in zygotes developing from spermatidfertilized oocytes. Anyway, these examples show,
first, that artificial manipulation of oocyte activation will be needed to increase the efficiency of
fertilization with sperm precursor cells and, second,
that the nature of this manipulation will depend
strictly on the stage of the sperm precursor cell
that is used. Further improvements to the success
rates of human fertilization using spermatids can
thus be expected to result from a better understanding of the relationship between the spermatid
developmental stage, nuclear maturity, the activity
and stability of the oocyte-activating factors, and
the time needed for spermatid nucleus exposure to
ooplasmic factors after the injection. Based on
Immature sperm and oocyte activation
this knowledge, it will hopefully be possible to
determine the optimal combination of spermatid
plasma membrane destabilization before injection,
degree of ooplasmic aspiration during the injection
and the timing of eventual auxiliary treatments to
boost oocyte activation (ionophore, electroactivation, etc.).
References
Abassi, Y.A. and Foltz, K.R. (1994) Tyrosine
phosphorylation of the egg receptor for sperm at
fertilization. Dev. Biol., 164, 430-433.
Ayabe, T., Kopf, G.S. and Schultz, R.M. (1995)
Regulation of mouse egg activation: presence of
ryanodine receptors and effects of microinjected
ryanodine and cyclic ADP ribose on uninseminated
and inseminated eggs. Development, 121, 2233-2244.
Bement, W.M. and Capco, D.G. (1991) Parallel pathways
of cell cycle control during Xenopus egg activation.
Proc. Natl. Acad. Sci. USA, 88, 5172-5176.
Berridge, M.J. (1996) Regulation of calcium spiking in
mammalian oocytes through a combination of inositol
trisphosphate-dependent entry and release. Mol. Hum.
Reprod., 2, 386-388.
Blobel, C.P., Wolfsberg, T.G., Turck, C.W. et al. (1992)
A potential fusion peptide and an integrin ligand
domain in a protein active in sperm-egg fusion.
Nature, 356, 248-252.
Borsuk, E. (1991) Anucleate fragments
of
parthenogenetic eggs and of maturing oocytes contain
complementary factors required for development of a
male pronucleus. Mol. Reprod. Dev., 29, 150-156.
Ciapa, B. and Epel, D. (1991) A rapid change in
phosphorylation on tyrosine accompanies fertilization
of sea urchin eggs. FEBS Lett., 293, 110-115.
Dale, B., DeFelice, L.J. and Ehrenstein, G. (1985)
Injection of a soluble sperm extract into sea urchin
eggs triggers the cortical reaction. Experientia, 41,
1068-1070.
Devroey, P., Liu, J., Nagy, Z. et al. (1995) Pregnancies
after testicular sperm extraction and intracytoplasmic
sperm injection in non-obstructive azoospermia. Hum.
Reprod., 10, 1457-1460.
Dozortsev, D., Rybouchkin, A., De Sutter, P. et al.
(1995 a) Human oocyte activation following
intracytoplasmic sperm injection: the role of the sperm
cell. Hum. Reprod., 10, 403^107.
Dozortsev, D., Rybouchkin, A., De Sutter, P. et al.
(1995b) Sperm plasma membrane damage prior to
intracytoplasmic sperm injection: a necessary
condition for sperm nucleus decondensation. Hum.
Reprod., 10, 2960-2964.
Eckberg, W.R. and Miller, A.L. (1995) Propagated
and nonpropagated calcium transients during egg
activation in the annelid, Chaetopterus. Dev. Biol,
172, 654-664.
Fishel, S., Green, S., Bishop, M. etal. (1995a) Pregnancy
after intracytoplasmic injection of spermatid. Lancet,
345, 1641-1642.
Fishel, S., Lisi, F., Rinaldi, L. et al. (1995b) Systematic
examination of immobilizing spermatozoa before
intracytoplasmic sperm injection in the human. Hum.
Reprod., 10, 497-500.
Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid
conception: a stage too early, or a time too soon?
Hum. Reprod., 11, 1371-1375.
Fissore, R.A., Pinto-Correia, C. and Robl, J.M. (1995)
Inositol trisphosphate-induced calcium release in the
generation of calcium oscillations in bovine eggs.
Biol. Reprod., 53, 166-11 A.
Foltz, K.R., Partin, J.S. and Lennarz, W.J. (1993) Sea
urchin egg receptor for sperm: sequence similarity of
binding domain and hsp70. Science, 259, 1421-1425.
Fusi, F.M., Vignali, M , Busacca, M. et al. (1992)
Evidence for the presence of an integrin cell adhesion
receptor on the oolemma of unfertilized human
oocytes. Mol. Reprod. Dev., 31, 215-222.
Gould, M. and Stephano, J.L. (1987) Electrical responses
of eggs to acrosomal protein similar to those induced
by sperm. Science, 235, 1654-1656.
Grandin, N. and Charbonneau, M. (1991) Intracellular
pH and intracellular free calcium responses to protein
kinase C activators and inhibitors in Xenopus eggs.
Development, 112, 461-470.
Homa, S.T. and Swann, K. (1994) A cytosolic sperm
factor triggers calcium oscillations and membrane
hyperpolarizations in human oocytes. Hum. Reprod.,
9, 2356-2361.
Hewitson, L.C., Simerly, C.R., Tengowski, M.W. et al.
(1996) Microtubule and chromatin configurations
during rhesus intracytoplasmic sperm injection:
successes and failures. Biol. Reprod., 55, 271-280.
In't Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995)
Sex chromosomal abnormalities and intracytoplasmic
sperm injection. Lancet, 346, 773.
Iwao, Y. and Fujimura, T. (1996) Activation of Xenopus
eggs by RGD-containing peptides accompanied by
intracellular Ca 2+ release. Dev. Biol, 111, 558-567.
Jaffe, L.A. (1990) First messengers at fertilization. J.
Reprod. Fertil Suppl, 42, 107-116.
Kimura, Y. and Yanagimachi, R. (1995a) Mouse oocytes
injected with testicular spermatozoa and round
spermatids can develop into normal offspring.
Development, 121, 2397-2405.
Kimura, Y. and Yanagimachi, R. (1995b) Development
of normal mice from oocytes injected with secondary
spermatocyte nuclei. Biol Reprod., 53, 855-862.
Kline, J.T. and Kline, D. (1994) Regulation of
intracellular calcium in the mouse egg: evidence for
inositol trisphosphate-induced calcium release, but125
not
J.Tesarik
calcium-induced calcium release. Biol. Reprod., 50,
193-203.
Lorca, T., Cruzalegni, F.H., Fesquet, D. et al. (1993).
Calmodulin-dependent protein kinase II mediates
inactivation of MPF and CSF upon fertilization of
Xenopus eggs. Nature, 366, 270-273.
Mansour, R.T., Aboulghar, M.A., Serour, G.I. et al
(1996) Successful intracytoplasmic sperm injection
without performing cytoplasmic aspiration. Fertil.
Steril, 66, 256-259.
Miyazaki, S. (1988) Inositol 1,4,5-trisphosphate-induced
calcium release and guanine nucleotide-binding
protein-mediated periodic calcium rises in golden
hamster eggs. J. Cell Biol, 106, 345-353.
Miyazaki, S. (1995) Inositol trisphosphate receptor
mediated spatiotemporal calcium signalling. Cum
Opin. Cell Biol, 7, 190-196.
Miyazaki, S., Katayama, Y. and Swann, K. (1990)
Synergistic activation by serotonin and GTP analogue
and inhibition by phorbol ester of cyclic Ca 2+ rises
in hamster eggs. J. Physiol, 426, 209-227.
Miyazaki, S., Shirakawa, H., Nakada, K. et al. (1993)
Essential role of the inositol 1,4,5-trisphosphate
receptor/Ca2+ release channel in Ca 2+ waves and
Ca 2+ oscillations at fertilization of mammalian eggs.
Dev. Biol, 158, 62-78.
Moore, G.D., Kopf, G.S. and Schultz, R.M. (1993)
Complete mouse egg activation in the absence of
sperm by stimulation of an exogenous G proteincoupled receptor. Dev. Biol, 159, 669-678.
Myles, D.G., Kimmel, L.H., Blobel, C.P. et al (1994)
Identification of a binding site in the disintegrin
domain of fertilin required for sperm-egg fusion.
Proc. Natl. Acad. Sci. USA, 91, 4195-4198.
Nagy, Z.P., Liu, J., Joris, H. et al. (1994) Time-course
of oocyte activation, pronucleus formation and
cleavage in human oocytes fertilized
by
intracytoplasmic sperm injection. Hum. Reprod., 9,
1743-1748.
Ogura, A., Matsuda, J. and Yanagimachi, R. (1994)
Birth of normal young after electrofusion of mouse
oocytes with round spermatids. Proc. Natl. Acad. Sci.
USA, 91, 7460-7462.
Ozil, J.P. and Swann, K. (1995) Stimulation of repetitive
calcium transients in mouse eggs. J. Physiol, 483,
331-346.
Palermo, G., Joris, H., Devroey, P. et al. (1992)
Pregnancies after intracytoplasmic injection of a single
spermatozoon into an oocyte. Lancet, 340, 17-18.
Palermo, G.D., Cohen, J., Alikani, M. et al. (1995)
Intracytoplasmic sperm injection: a novel treatment
for all forms of male factor infertility. Fertil Steril,
63, 1231-1240.
Palermo, G.D., Schlegel, P.N., Colombero, L.T. et al
(1996) Aggressive sperm immobilization prior to
intracytoplasmic sperm injection with immature
126
spermatozoa improves fertilization and pregnancy
rates. Hum. Reprod., 11, 1023-1029.
Parrington, J., Swann, K., Shevchenko, V.I. et al. (1996)
Calcium oscillations in mammalian eggs triggered by
a soluble sperm protein. Nature, 379, 364-368.
Primakoff, P., Hyatt, H. and Tredick-Kline, J. (1987)
Identification and purification of a sperm surface
protein with a potential role in sperm-egg membrane
fusion. J. Cell Biol, 104, 141-149.
Shankar, G., Davison, I., Helfrich, M.H. et al (1993)
Integrin
receptor-mediated
mobilization
of
intracellular calcium in rat osteoclasts. J. Cell Sci.,
105, 61-68.
Shilling, KM., Carroll, D.J., Muslin, A.J. et al (1994)
Evidence for both tyrosine kinase and G-proteincoupled pathways leading to starfish egg activation.
Dev. Biol, 162, 590-599.
Silber, S.J. (1994) The use of epididymal sperm in
assisted reproduction. In Tesarik, J. (ed.), Male Factor
in Human Infertility. Ares-Serono Symposia, Rome,
pp. 335-368.
Sousa, M. and Tesarik, J. (1994) Ultrastructural analysis
of fertilization failure after intracytoplasmic sperm
injection. Hum. Reprod., 9, 2374-2380.
Sousa, M., Barros, A. and Tesarik, J. (1996a) The
role of ryanodine-sensitive Ca 2+ stores in the Ca 2+ oscillation machine of human oocytes. Mol Hum.
Reprod., 2, 265-272.
Sousa, M., Barros, A. and Tesarik, J. (1996b)
Developmental changes in calcium dynamics, protein
kinase C distribution and endoplasmic reticulum
organization in human preimplantation embryos. Mol.
Hum. Reprod., 2, 967-977.
Sousa, M., Barros, A., Mendoza, C. and Tesarik, J.
(1996c) Effects of protein kinase C activation and
inhibition on sperm-, thimerosal-, and ryanodineinduced calcium responses of human oocytes. Mol.
Hum. Reprod., 2, 699-708.
Sousa, M., Mendoza, C , Barros, A. and Tesarik, J.
(1996d) Calcium responses of human oocytes after
intracytoplasmic
injection
of
leukocytes,
spermatocytes and round spermatids. Mol. Hum.
Reprod., 2, 853-857.
Strieker, S.A. (1996) Repetitive calcium waves induced
by fertilization in the nemertean worm Cerebratulus
lacteus. Dev. Biol, 176, 243-263.
Swann, K. (1990) A cytosolic sperm factor stimulates
repetitive calcium increases and mimics fertilization
in hamster eggs. Development, 110, 1295-1302.
Swann, K. (1992) Different triggers for calcium
oscillations in mouse eggs involve a ryanodinesensitive Ca 2+ store. Biochem. J., 287, 79-84.
Swann, K. (1994) Ca 2+ oscillations and sensitization of
Ca 2+ release in unfertilized mouse eggs injected with
a sperm factor. Cell Calcium, 15, 331-339.
Swann, K. and Ozil, J.P. (1994) The dynamics of the
Immature sperm and oocyte activation
calcium signal that triggers mammalian egg activation.
Int. Rev. Cytol, 152, 182-222.
Swann, K., Igusa, Y. and Miyazaki, S. (1989) Evidence
for an inhibitory effect of protein kinase C on Gprotein-mediated repetitive calcium transients in
hamster eggs. EMBO J., 8, 3711-3718.
Tarone, G., Russo, M.A., Hirsch, E. et al. (1993)
Expression of bj integrin complexes on the surface
of unfertilized mouse oocyte. Development, 117,
1369-1375.
Taylor, C.T., Lawrence, Y.M., Kingsland, n. et al. (1993)
Oscillations in intracellular free calcium induced by
spermatozoa in human oocytes at fertilization. Hum.
Reprod., 8, 2174-2179.
Tesarik, J. (1995) Sex chromosome abnormalities after
intracytoplasmic sperm injection. Lancet, 346, 1096.
Tesarik, J. (1996a) Use of immature germ cells for the
treatment of human infertility. In Van Steirteghem,
A., Devroey, P. and Tournaye, H. (eds), Male Infertility.
Bailliere Tindall, London.
Tesarik, J. (1996b) Fertilization of oocytes by injecting
spermatozoa, spermatids and spermatocytes. Rev.
Reprod., 1, 149-152.
Tesarik, J. and Mendoza, C. (1996) Spermatid injection
into human oocytes. I. Laboratory techniques and
special features of zygote development. Hum. Reprod.,
11, 772-779.
Tesarik, J. and Sousa, M. (1994) Comparison of Ca 2+
responses in human oocytes fertilized by subzonal
insemination and by intracytoplasmic sperm injection.
Fertil. Steril, 62, 1197-1204.
Tesarik, J. and Sousa, M. (1995a) More than 90%
fertilization rates after intracytoplasmic sperm
injection and artificial induction of oocyte activation
with calcium ionophore. Fertil. Steril, 63, 343-349.
Tesarik, J. and Sousa M. (1995b) Key elements of
a highly efficient intracytoplasmic sperm injection
technique: Ca 2+ fluxes and oocyte cytoplasmic
dislocation. Fertil. Steril, 64, 770-776.
Tesarik, J. and Sousa, M. (1996) Mechanism of calcium
oscillations in human oocytes: a two-store model.
Mol Hum. Reprod., 2, 383-390.
Tesarik, J. and Testart, J. (1994) Treatment of sperminjected human oocytes with Ca 2+ ionophore supports
the development of Ca 2+ oscillations. Biol. Reprod.,
51, 385-391.
Tesarik, J., Sousa, M. and Testart, J. (1994) Human
oocyte activation after intracytoplasmic sperm
injection. Hum. Reprod., 9, 511-518.
Tesarik, J., Mendoza, C. and Testart, J. (1995a) Viable
embryos from injection of round spermatids into
oocytes. N. Engl. J. Med., 333, 525.
Tesarik, J., Sousa, M. and Mendoza, C. (1995b) Sperminduced calcium oscillations of human oocytes show
distinct features in oocyte center and periphery. Mol.
Reprod. Dev., 41, 257-263.
Tesarik. J., Rolet, E, Brami, C. et al. (1996) Spermatid
injection into human oocytes. II. Clinical application
in the treatment of infertility due to non-obstructive
azoospermia. Hum. Reprod., 11, 780-783.
Van den Bergh, A., Bertrand, E., Biramane, J. et al.
(1995) Importance of breaking a spermatozoon's tail
before intracytoplasmic injection: a prospective
randomized trial. Hum. Reprod., 10, 2819-2820.
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993)
High fertilization and implantation rates after ICSI.
Hum. Reprod., 8, 1061-1066.
Vitullo, A.D. and Ozil, J.P. (1992) Repetitive calcium
stimuli drive meiotic resumption and pronuclear
development during mouse oocyte activation. Dev.
Biol, 151, 128-136.
Watanabe, N., Hunt, T., Ikawa, Y. et al. (1991)
Independent inactivation of MPF and cytostatic factor
(Mos) upon fertilization of Xenopus eggs. Nature,
352, 246-248.
Wolfsberg, T.G., Bazan, J.J., Blobel, C.P. et al. (1993)
The precursor region of a protein active in spermegg fusion contains a metalloprotease and a disintegrin
domain: structural, functional and evolutionary
implications. Proc. Natl. Acad. Sci. USA, 90,
10783-10787.
Yamada, K.M. and Miyamoto, S. (1995) Integrin
transmembrane signalling and cytoskeletal control.
Curr. Opin. Cell Biol, 7, 681-689.
Yue, C , White, K.L., Reed, W.A. et al. (1995) The
existence of inositol 1,4,5-trisphosphate and ryanodine
receptors in mature bovine oocytes. Development,
121, 2645-2654.
127