Human Reproduction Update, Vol.10, No.1 pp. 53±65, 2004
DOI: 10.1093/humupd/dmh006
Electrical events during gamete maturation and
fertilization in animals and humans
Elisabetta Tosti1,3 and Raffaele Boni2
1
Laboratory of Cell Biology, Stazione Zoologica, Villa Comunale, 80121 Naples and 2Department of Animal Science, University of
Basilicata, Potenza, Italy
To whom correspondence should be addressed: E-mail: [email protected]
Gamete cells are electrogenic, i.e. capable of responding to electrical stimuli and modifying their electrical properties during the crucial periods of maturation and fertilization. Ion channels have been widely demonstrated on the
plasma membrane of the oocyte and spermatozoon in all animals studied, and electrical modi®cations in gametes
are due to ion currents that are modulated via these ion channels. The modi®cation of intracellular calcium levels in
gametes has been extensively studied, and these modi®cations are recognized to be a second messenger system for
gamete maturation and fertilization. Other ions also move through the plasma membrane, either in association with
or independent of calcium, and these generate typical features such as fertilization currents and oscillation of resting
potential. These modi®cations were ®rst studied in marine invertebrates, and the observations subsequently compared with mammalian systems, including human. The precise role played by these currents in the processes of maturation and fertilization is still poorly understood; however, recent research opens new frontiers for their clinical
and technological application.
Key words: electrophysiology/fertilization/gamete maturation/ion channels/ion currents
Introduction
The growth of germinal cells follows unique rhythms and
dynamics that differ from those of somatic cells. A preliminary
long-lasting quiescent phase allows the proper development of
cellular growth and differentiation, and mechanisms of gamete
maturation then follow at a tumultuous cascading speed. Gamete
metabolism is then newly modi®ed during the process of
fertilization, and the success of the process relies upon a reciprocal
activation of the male and female cells. Speci®cally, sperm
competence for activation and fusion is triggered by the external
investments of the oocyte, and the activated spermatozoon then
drives the oocyte into metabolic activation. Electrical modi®cations of the plasma membrane underlie both of these processes.
Electrical events are one of the ®rst indications that gametes are
undergoing activation steps during maturation and fertilization
(Moreau et al., 1985; Dale, 1994; Darszon et al., 1999). This is due
to activation of ion channels located on the plasma membrane. An
exchange of ions between the internal and the external compartments of the cell generates an electrical current, and this modi®es
the electrical steady state of the cell. Fertilization is the best
example of the link between a transient electrical modi®cation and
a new metabolic condition within a cell. A species-dependent
fertilization current and a large hyperpolarization or depolarization
of membrane potential occur in the oocyte shortly after sperm
entry. These events coincide with the initiation of embryo
development.
In this review, we focus on the electrical properties of the
plasma membrane of animal germ cells, i.e. the oocyte and the
spermatozoon, throughout their maturation and following their
union to form a new individual. After a preliminary description of
the main parameters used for electrophysiological study of the cell
plasma membrane, we report information on the properties and
changes in intracellular ions, ion channels and ion currents that
occur during gamete maturation and fertilization.
Electrophysiology of the cell
The plasma membrane marks the border between the internal and
external compartments of a cell. The ¯uids in these compartments
consist of saline solutions of differing compositions. The intracellular ¯uid contains organelles bathed in a matrix whose ion
content differs from that of the external compartment. This
compartmentalization must be maintained, and is essential for cell
activity as well as for cell viability; it is regulated by speci®c
dynamics that are modulated by complex mechanisms, as
described below.
Potassium cations (K+) are the most signi®cant intracellular
ions, whereas sodium (Na+) and calcium (Ca2+) cations are
signi®cant extracellularly. Chloride anion (Cl±) is present at higher
Human Reproduction Update Vol. 10 No. 1 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
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3
E.Tosti and R.Boni
54
The patch-clamp technique, ®rst described by Neher and
Sakmann (1976), is the most popular technique used to study
cell currents; the basic con®gurations of this technique involve
whole cell or single channel recordings. The former measures
currents from an entire cell under voltage clamp and this is
normally obtained after seal and destruction of the patched
membrane. The single channel con®guration records a channel
activity inside the intact patched membrane. The application of
Giga-seal resistance and different con®gurations of the patchclamp technique literally revolutionized the study of membrane
electrophysiology, and allowed access to new information about
cell function (for review see: Neher, 1988, 1992; Neher and
Sakmann, 1992; Levis and Rae, 1998). Figure 1 lists a glossary of
the main electrophysiological parameters described above.
Electrical properties of the gamete plasma membrane
The oocyte
Oocytes are electrogenic cells that block their development during
different species-speci®c stages of the meiotic cycle. Early studies
on the role of ions in oocyte physiology date back to 1946, when
Edward Chambers started to investigate Na+ and K+ exchange
during fertilization in the sea urchin (for review see Chambers,
1989a).
Different species of echinoderms, i.e. star®sh and sea urchin,
have been used extensively to study the electrophysiological
properties of the oocyte plasma membrane. In star®sh, immature
oocytes exhibit three types of voltage-dependent currents: an
inward Ca2+, a fast transient K+ and an inwardly rectifying K+
current (Moody and Lansman, 1983). During hormone-induced
in vitro maturation, a gradual change in the amplitude of all three
currents is seen: the Ca2+ current becomes larger whereas both K+
currents become smaller (Moody and Lansman, 1983). These
changes are also associated with a decrease in membrane
conductance and a depolarization of RP during maturation due
to changes in Na+ and K+ conductance (Miyazaki et al., 1975a,b;
Moreau and Cheval, 1976). The sea urchin is the species that has
been most extensively studied for mechanisms of fertilization (for
review see Monroy, 1986). However, the electrical properties of its
plasma membrane have not been clearly elucidated, possibly due
to the fact that patching the oocyte plasma membrane in this
species is technically dif®cult to accomplish. Earlier studies on sea
urchin oocyte RP suggested that the membrane has a high
permeability to K+ and a lower permeability to Na+ and Cl±
(Steinhardt et al., 1971; Jaffe and Robinson, 1978). The presence
of Ca2+ channels was ®rst demonstrated in a comparative study by
Okamoto et al. (1977). Electrical studies of oocytes during the
germinal vesicle breakdown (GVBD) stage suggest that immature
oocytes have a high K+ permeability which is lost during
maturation (Dale and De Santis, 1981).
Another marine invertebrate, the ascidian, has also been used to
study gamete biology. In oocytes of Boltenia villosa, Block and
Moody (1987) described three principal voltage-dependent currents as follows: (i) a transient inward Na+ current, (ii) a transient
inward Ca2+ current, and (iii) an inwardly rectifying K+ current. A
more accurate characterization of Ca2+ channel sub-types performed in Ciona intestinalis oocytes showed the presence of Ltype Ca2+ currents (Dale et al., 1991), suggesting that this calcium
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concentration externally, whereas there is a great variation in the
anions present inside the cell. These intracellular anions include
negative charges provided by several constituents that carry
phosphate and carboxyl groups. The different distribution of
electrical charges inside and outside the cell creates an electrical
gradient across the membrane, known as voltage, and this is
measured in Volts. The voltage difference across the cell
membrane creates a store of potential energy in the form of an
ion gradient, giving rise to a transmembrane potential known as
resting potential (RP). This parameter changes in relation to ion
distribution and membrane permeability.
In the majority of cells studied, the RP is negative, in a range of
±10 to ±100 mV (Hagiwara and Jaffe, 1979). Potassium is
electively stored inside the cell, and it is the ion that contributes
mainly to determining and regulating the resting potential (De
Felice, 1997). The K+ gradient and differences in membrane ion
permeability are, in turn, determined by speci®c transport proteins
and ion channels in the plasma membrane. The transport proteins
(ion pumps) maintain the concentration gradients that determine
resting potential (e.g. Na+/K+ pump) and the general homeostasis
of the cell. Ion channels are characterized by their speci®city,
gating, conductance and sensitivity to drugs, and these act as gated
ion-selective pores that allow ions to move down their concentration gradients. An ion channel is speci®c if it allows the passage
of one ion species predominantly. Ion channel opening is triggered
in response to (i) a ligand (second messenger-operated channels)
(Sutcliffe et al., 1998), (ii) a change in voltage (voltage-operated
channels) (Terlau and Stuhmer, 1998) or (iii) a mechanical
stimulus (stretch-activated channels) (Hamill and McBride, 1996;
Saitou et al., 2000). Moreover, each channel has a proper
conductance, which is measured in Siemens (S). A speci®c ion
channel shows a mean conductance between 5 and 10 pS;
however, some highly speci®c channels, such as Ca2+ channels,
may have a conductance that is <1 pS. Non-speci®c channels show
higher conductance measurements. The passage of ions across
membrane ion channels determines an electrical ¯ux which is
called an ion current; this is measured in Amperes (A). Normally,
the ion current is associated with a change in RP, since the
concentration of ions inside the cell is transiently modi®ed. In
particular, depolarization of the plasma membrane causes a shift of
RP towards more positive values, whereas hyperpolarization
modi®es the RP towards more negative values. The type and the
number of channels within a cell depends on several parameters
which determine the grade of cell excitability. Electrical excitability is an essential property of neurons, and this involves
characteristic sets of voltage-dependent ion channels. Muscle cells
(Katz, 1966), germ cells (Chambers, 1989a), many endocrine cells
(Ozawa and Sand, 1986) and even so-called non-excitable cells,
such as glial cells (Barres et al., 1990), express voltage-dependent
ion channels at a certain stage of cellular differentiation. Hence,
electrical properties characterize individual cells in relation to
their function and differentiation (for review see Takahashi and
Okamura, 1998).
Capacitance is another example of an electrophysiological
parameter which every cell has by virtue of the membrane lipid
bilayer. This is well described in the sea urchin (McCulloh and
Chambers, 1992) and is also reported in mouse oocytes (Lee et al.,
2001), where an increase in plasma membrane capacitance as well
as cortical granule exocytosis follows sperm±oocyte fusion.
Ion channels in gametes
current component has a role in regulating cytosolic calcium
during early developmental processes.
Schlichter (1989a) has provided an excellent overview on ion
channels in amphibian oocytes; a role for Cl± currents emerges in
both immature and mature oocytes in all the species studied. In the
European frog Rana esculenta, changes in membrane permeability
occur during maturation; in particular, K+ and Cl± voltage-gated
currents present in the immature oocytes disappear in mature
oocytes, replaced by a Na+ current (Taglietti et al., 1984). A Ca2+dependent Cl± current has been shown in Xenopus immature
oocytes (Barish, 1983), and Na+ currents similar to those of
neuronal cells, as well as other non-speci®c ion currents, have also
been described (Bourinet et al., 1992; Weber, 1999). The same
currents are present in immature metaphase I oocytes of R. pipiens,
each contributing differently to subsequent fertilization potential
(Schlichter, 1989b); in particular, Cl± is lost as the oocyte
undergoes maturation (Schlichter, 1983). A noticeable voltagedependent hydrogen current was ®rst demonstrated in the axolotl
Ambistoma oocytes (Barish and Baud, 1984).
In addition to the above species, ion currents that play a possible
role in the mechanism of maturation and fertilization have been
observed in other non-mammalian species such as molluscs
(Moreau et al., 1996; Ouadid-Ahidouch, 1998; Gould et al., 2001;
E.Tosti, unpublished data), and marine polychaetes (Gunning,
1983; Fox and Krasne, 1984).
In mammals, separating the oocyte from the cumulus investment makes the measurement of electrical parameters technically
dif®cult, and therefore there are very few papers that discuss
electrical properties of the mammalian oocyte plasma membrane
during maturation. A functional separation between these two
units occurs spontaneously in the mature oocyte, but during
immature stages there is an intimate interaction between the
oocyte and the cumulus cells, and this is maintained by
intercellular communications such as gap junctions (Gilula et al.,
1978; Canipari, 2000). Notwithstanding this interaction, cumulus
cells and oocytes do maintain different membrane potentials
(Emery et al., 2001). The signi®cance of this ®nding is still
unclear, especially if it is related to the high level of electrical
coupling found between these two cell types and to the presence of
low resistance channels such as gap junctions (Furshpan and
Potter, 1959). The cumulus cells are devoted to transmitting
stimuli to the oocyte, such as those involved in maintaining (Aktas
et al., 1995) or removing (Batta and Knudsen, 1980; Mattioli et al.,
1990) meiotic arrest. In the former case, the oocyte RP is
maintained under hyperpolarizing conditions, whereas in the latter
case a depolarization occurs as consequence of the action of
gonadotrophins on the cumulus cells (Mattioli et al., 1990, 1991),
leading to progression of meiosis. Membrane properties of
cumulus cells may play an interesting role in follicle and oocyte
growth and maturation (Mattioli et al., 1993). Depolarization of
granulosa cells was, in fact, addressed as an essential feature of
follicular maturation; this hypothesis is supported by the ®nding
that differential granulosa cell proliferation, steroidogenic capability and apoptosis can be in¯uenced by selective antagonism of
granulosa cell K+ channels with distinct molecular correlates,
electrophysiological properties, and expression patterns
(Manikkam et al., 2002).
This coupling decreases at the end of maturation, along with
cumulus expansion and the breaking of heterologous gap junction
communications (Gilula et al., 1978; Suzuki et al., 2000).
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Figure 1. A schematic representation of electrophysiological parameters. Voltage is generated by the differential distribution of positive and negative ions
across the cell membrane. The resting potential is the separation of charge, which generates the voltage difference across the cell membrane. This potential
energy is stored as an ion gradient, i.e. different concentrations of ions inside and outside the cell. Ion channels are speci®c proteins located within the lipid
bilayer that allow the passage of speci®c ions through the plasma membrane. Ion current is the movement of charges determined by the passage of ions through
ion channels.
E.Tosti and R.Boni
56
these studies, a large variability was found in mitochondrial
membrane potential, and this has been considered as a discriminating parameter for evaluating oocyte quality (Van Blerkom
et al., 2002; Wilding et al., 2003).
The role played by membrane potential modi®cations in oocyte
maturation is still unclear. The change in current amplitude,
density and membrane capacitance in the oocytes of all animals
described appears to be related to progression of meiotic
maturation and to the preparation of the oocyte for fertilization.
In fact, most of the oocytes studied to date show a clear change in
their electrical properties upon fertilization. The oocyte hyperpolarization occurring at the beginning of the maturation process is
due to a selective permeability to K+ ions (Powers and Tupper,
1977). However, maturation is arrested by the K+-selective
ionophore, valinomycin (Powers and Biggers, 1976). In addition,
the depolarization of immature oocytes following LH exposure
(Dawson and Conrad, 1972; Georgiou et al., 1984; Kline, 1988)
follows granulosa cell depolarization and may, hence, represent a
consequence rather than a cause of the meiotic block removal.
The spermatozoon
The role of the fertilizing spermatozoon is to transport the male
genome into the oocyte and to trigger the oocyte into metabolic
activation. In order to become competent for oocyte activation, the
spermatozoon must ®rst undergo a series of modi®cations induced
by contact with components of the external layers of the oocyte.
Many of these processes involve a change in the electrical
properties of the plasma membrane. During the past decade, an
improvement in techniques of investigation such as voltage- and
ion-sensitive ¯uorescent indicators, immunocytochemistry,
pharmacology and DNA recombinant technology has led to an
accumulation of evidence that increasingly implies a role for ion
channels in sperm physiology (Darszon et al., 1999; 2001, 2002).
The application of the patch-clamp technique to monolayers
generated from a mixture of lipid vesicles and isolated sperm
membranes had a signi®cant technological impact on our understanding of the electrophysiology of the sperm cell. Single channel
recording from sea urchin sperm plasma membrane identi®ed the
presence of K+ and Cl± channel activity (LieÁvano et al., 1985).
Subsequently, this was extended to a series of animal models
revealing the presence of several types of ion channels on the
sperm plasma membrane, including cation (K+ and Ca2+) and
anion (Cl±) channels (Chan et al., 1997). However, the molecular
mechanisms that link the action of ion channels to gamete
physiology are yet to be clari®ed.
Immature germ cells offer a technical advantage, in that their
size and immobility allow the patch-clamp technique to be easily
applied. Although only a few studies have so far been conducted
on the role of ion channels in immature male germ cells, they have
supplied useful information about the role of ion channels in sperm
physiology. These may only be extrapolated to mature sperm if it
is assumed that immature germ cells express the same proteins that
are present on the sperm plasma membrane.
The presence of K+ and Ca2+ currents in rat spermatogenetic
cells was ®rst demonstrated by Hagiwara and Kawa (1984).
Subsequently, a more accurate analysis showed that rat spermatids
exhibited a negative RP, determined by Cl± and K+ conductance
with a minor contribution of Na+ conductance (Reyes et al., 1994).
A role for Cl± conductance in spermatogenesis was later shown in
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However, this is not accompanied by a simultaneous decrease in
electrical coupling (Racowsky and Satterlie, 1985, 1987). The
expansion of cumulus in the mature oocyte causes a depolarization
of the resting potential which reaches the same value as that
obtained in immature oocytes after cumulus removal (Emery et al.,
2001). The arti®cial separation of these two integrated components
may therefore generate artefacts. On the other hand, the maintenance of the cumulus±oocyte complex prevents advanced investigation of the plasma membrane, which can only be performed after
cumulus removal. In our experience with bovine oocytes, we did
not ®nd signi®cant differences between immature (GV stage) and
in vitro matured (MII stage) oocytes with regards to the RP values;
this could be attributed to cumulus removal. However, RP
variations were found during meiosis progression (Tosti et al.,
2000). In addition, the removal of cumulus cells allowed us to
appreciate large variations in plasma membrane ion channels,
related both to conductance as well as to Ca2+ stores (Boni et al.,
2002; Tosti et al., 2002).
A wide range of modi®cations were found on the oocyte plasma
membrane in association with rapid modi®cations of the intracellular environment, such as changes in free Ca2+ concentration.
These modi®cations occur both on resumption of meiotic arrest
due to hormonal signals (Eppig et al, 1984) and on sperm entry
(Miyazaki and Igusa, 1981b). In the immature oocyte, plasma
membrane properties vary according to meiosis progression
(McCulloh and Levitan, 1987; Tosti et al., 2000) as well as
according to developmental competence (Murnane and De Felice,
1993; Boni et al., 2002). In particular, in bovine oocytes the RP
depolarizes during meiosis progression, after a preliminary
hyperpolarization at GVBD stage (Tosti et al., 2000). The opposite
pattern has also been recorded in relation to plasma membrane
permeability (Murnane and De Felice, 1993; Tosti et al., 2000).
Biophysical and pharmacological evidence strongly suggests that
these electrical currents on the oocyte plasma membrane represent
L-type Ca2+ channels. These channels have been demonstrated to
underlie meiosis resumption in ascidian (Dale et al., 1991), mussel
(Tomkoviak et al, 1997), Pleurodeles (Ouadid-Ahidouch, 1998)
and mouse (Murnane et al., 1988) oocytes. In bovine oocytes, the
activity of the L-type Ca2+ channels decreases throughout meiosis
progression (Tosti et al., 2000). Since Ca2+ is necessary for meiosis
progression (Homa, 1991, 1995; He et al., 1997), this pattern may
support the cytosolic Ca2+ rise in GV following LH and/or growth
factor exposure (Mattioli et al., 1998; Hill et al., 1999). This
highlights the additional role of external Ca2+ in mobilization of
intracellular Ca2+ during oocyte activation and fertilization in this
species.
In humans, the ®rst studies on membrane potential were
performed by measuring intracellular electrical recordings in
immature oocytes collected by ovariectomy (Eusebi et al., 1984;
Dolci et al., 1985). Using the same electrophysiological technique,
Feichtinger et al. (1988) tested membrane potential variations in
oocytes collected at different stages of maturation by ultrasoundguided retrieval. De Felice et al. (1988) ®rst applied patch-clamp
and whole cell recordings to immature and mature oocytes. The
most frequently observed channel in mature oocytes was a 60 pS
non-inactivating, K+-selective pore, which was activated by
depolarization. Recently, the recording of membrane potential
has been extended to oocyte mitochondrial membranes by using
¯uorescent probes combined with image analysis techniques. In
Ion channels in gametes
Chemotaxis
Chemotaxis is described by the activation of sperm motility and
the attraction of activated sperm towards the oocyte. The
communication of pre-contact gametes by means of chemotaxis
is well documented in species with external fertilization (for
review see Tosti, 1994). Rothschild (1948) demonstrated that
extreme conditions of K+ concentration, pH and oxygen tension
maintain sea urchin sperm quiescent in the testis. After spawning,
a change in these physical parameters induced sperm to swim, by
acting on the tail axonema. Subsequent studies demonstrated that
sperm motility is triggered by environmental cues, including
diffusible compounds from the outer layer of oocytes, through
transduction events involving sperm ion channels (Morisawa,
1994). In particular, many small peptides on the external envelope
of echinoderm oocytes can induce ion ¯uxes causing a mobilization of second messengers. All of these peptides have a similar
function in inducing sperm motility; they essentially use K+
channel modulation as the ®rst response to their binding with a
receptor on the sperm plasma membrane (LieÁvano et al., 1985; Lee
and Garbers, 1986). At spawning, K+ ef¯ux through this channel
causes a cascade of consecutive electrical events that can be
summarized as follows: hyperpolarization of the RP, Na+/H+
exchange, pH rise, increase in cyclic nucleotides, Na+ in¯ux,
depolarization of RP and Ca2+ ef¯ux (for review see Darszon et al.,
2001). In addition, the hyperpolarization is also strictly related to a
cation channel which has been found mainly in the ¯agellum and
whose function may be involved in ¯agellar beating (Gauss, 1998).
Chemotaxis in ascidians has been well described for some time
(Miller, 1975); however, the involvement of ion channels in this
process has been highlighted only recently by Morisawa's team
(Izumi et al., 1999), who proposed a mechanism similar to that
described above. In brief, an increase in K+ permeability occurs at
the point of contact of the sperm with the oocyte investment, and
this induces ±50 mV hyperpolarization of the sperm plasma
membrane which in turn elevates cAMP. A cascade of cAMPdependent kinases may then activate sperm motility (Izumi et al.,
1999). Moreover, T- type Ca2+ channels were found to be related
to the elevation of cAMP (Yoshida et al., 1994), suggesting a role
for Ca2+ in ascidian sperm chemotaxis. In contrast to these
®ndings, recent evidence showed that ¯agellar movements in
ascidian sperm are regulated by a capacitative and not a voltagegated Ca2+ entry (Yoshida et al., 2003).
Osmolarity is one the main parameters that regulate sperm
motility in ®shes. In particular, a change in external osmolarity
seems to be converted into a change in intracellular K+ concentration. Subsequently, a K+ ef¯ux and intracellular Ca2+ rise has
been shown to act as a trigger that initiates sperm motility in
marine and freshwater teleosts, salmonid and rainbow trout
(Tanimoto and Morisawa, 1988; Oda and Morisawa, 1993;
Tanimoto et al., 1994; Takai and Morisawa, 1995).
In mammals, the role of follicular factors causing chemotactic
sperm attraction has only recently been highlighted (Eisenbach,
1999). However, the molecular mechanism underlying the process
is still poorly understood. Whereas the role of ion channels has
been clearly demonstrated in chemotaxis in invertebrates, in
mammals there is some evidence to indicate that intracellular Ca2+
release from stores in the mid-piece is involved. The Ca2+ increase
seems to mediate ¯agellar beating and, hence, chemotactic
response (Cook et al., 1994; Ho and Suarez, 2001; Suarez and
Ho, 2003).
Acrosome reaction
The acrosome reaction (AR) is the last activating event in the
spermatozoon as it becomes competent for fertilization. The
exocytosis of the acrosome and the consequent release of its
contained enzymes allows the spermatozoon to penetrate the
extracellular oocyte investments (Garbers, 1989). As with
chemotaxis, AR is induced at the contact with the outer layer of
the oocyte. Different compounds have been shown to be responsible for AR induction.
In echinoderms, sea urchin and star®sh, substances on the
oocyte jelly, such as fucose sulphate polymers (Alves et al., 1998),
sulphated fucose, galactose, xylose and the acrosome reactioninducing substance (ARIS), are involved in AR induction (Ikadai
and Hoshi, 1981; Alves et al., 1997; Koyota et al., 1997). The AR
takes place after binding between these substances and a speci®c
receptor on the sperm plasma membrane. The AR is recognized to
be an ion channel-regulated event; in fact, ion channels on the
sperm plasma membrane are mobilized within a few seconds of
sperm binding. Ca2+ in¯ux is an absolute requirement for AR in the
sperm of all species (Darszon et al., 1999), since it seems to be
involved in the dehiscence of the acrosomal vesicle and in
membrane fusion (Darszon et al., 2001). Along with Ca2+, K+ also
plays a role in the AR; this is supported by experimental evidence:
(i) K+ and Ca2+ channel inhibitors block the AR (Kazazoglou et al.,
1985; Yanagimachi, 1994) and (ii) speci®c Ca2+ and K+
ionophores trigger the AR (Collins and Epel, 1977; Schackmann
et al., 1978; Yanagimachi, 1994). In sea urchin the electrical
events underlying the AR after contact of oocyte jelly with a sperm
receptor result in an immediate Na+ and Ca2+ in¯ux and H+ and K+
ef¯ux. These events also result in a change in RP, an increase in
pH due to a Na+/H+ exchange and the intracellular Ca2+ rise
(Darszon et al., 1999). Evidence for the presence of two different
57
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Caenorhabditis elegans by Machaca et al. (1996); in this study a
new inwardly rectifying Cl± channel was characterized and put
forward as a candidate factor in spermatid differentiation.
In mouse spermatogenetic cells (Santi et al., 1998), a pHdependent Ca2+ permeability factor and a series of K+-selective
currents have been correlated with the state and function of mature
sperm. In particular, Ca2+ in¯ux is required for initiating the
acrosome reaction in mature sperm, whereas K+ seems to
contribute to the hyperpolarization and regulation of sperm
fertilizing capability (Munoz-Garay et al., 2001; Felix et al.,
2002). Recently, biomolecular studies provided evidence for the
regulation of T-type Ca2+ channel expression during mouse
spermatogenesis (Son et al., 2002) and suggested that these
sperm channels are implicated in the fertility block caused by
contraceptive substances (Bai and Shi, 2002a).
The above literature indicates a clear involvement of ion
channels in spermatogenesis and in the functionality of the mature
spermatozoon. More information is available on the electrical
properties of the spermatozoon immediately after ejaculation,
when it is not yet competent for fertilization. Fertilizing ability
relies on a ®nal maturation process known as sperm activation (or
capacitation in mammals), a process that may be divided into two
main events: chemotaxis (and hyperactivated motility) and the
acrosome reaction.
E.Tosti and R.Boni
Ca2+ channels and their synergistic action in inducing AR has been
reported by Guerrero and Darszon (1989). The Na+/H+ exchange
and pH rise is induced by mobilization of K+ channels that results
in a fast and transient hyperpolarization followed by a Ca2+mediated depolarization (LieÁvano et al., 1985; GonzaÁlez-MartõÁnez
and Darszon, 1987; GonzaÁlez-MartõÁnez et al., 1992). Cl±-selective
anion channels have also been identi®ed in sea urchin sperm
plasma membrane; they may have a role in the AR, in¯uencing the
RP of the gamete (Morales et al., 1993).
Mammalian sperm acquire the ability to fertilize at the end of a
process called `capacitation' (for review, see Yanagimachi, 1994).
This enables the spermatozoon to induce its AR, in response to the
zona pellucida (ZP) or to agents such as progesterone (Osman
et al., 1989). As in invertebrates, the contact of the sperm receptor
with the oocyte envelope (ZP in mammals) or AR inducers causes
elevation of intracellular Ca2+, pH increase and a change in the RP
(Arnoult et al., 1999; Florman et al., 1998; Patrat et al., 2000;
Darszon et al., 2001).
Ion channels on the head of the mammalian spermatozoa that
are responsible for Ca2+ entry and intracellular Ca2+ rise include:
(i) low and high voltage-activated channels, (ii) receptor-operated
Ca2+ channels and (iii) store-operated Ca2+ channels (Benoff,
1998). T-type voltage-gated Ca2+ channels have a pivotal role in
mediating the AR (Florman et al., 1998; Darszon et al., 1999). The
gating of these channels has been demonstrated in mammalian
(Arnoult et al., 1996; Publicover and Barratt, 1999) and in human
sperm by AR inducers such as progesterone (Garcia and Meizel,
1999) and mannose±bovine serum albumin (Blackmore and
Eisoldt, 1999; Son et al., 2000). Although the existence of
58
voltage-gated Ca2+ channels has been demonstrated in human
sperm (Linares-HernaÂndez et al., 1998), their function in the
induction of the AR has not yet been elucidated (for review see
Jagannathan et al., 2002). A capacitating Ca2+ entry mechanism
has been proposed as a possible mechanism for gating plasma
membrane Ca2+ channels. In mouse sperm, the existence of a Ca2+
in¯ux dependent on depletion of Ca2+ stores has recently been
shown (O'Toole et al., 2000). Rossato et al. (2001) have proposed
an interesting model to explain a possible mechanism for the AR in
human sperm. They demonstrated the existence of Ca2+ stores
whose depletion activates a double process: (i) gating of Ca2+activated K+ channels, with K+ ef¯ux causing a hyperpolarization,
and (ii) the capacitative gating of voltage-gated Ca2+ channels,
with a subsequent depolarization of the plasma membrane.
Anions have also been implicated in the mammalian AR
(Morales et al., 1993). This hypothesis was recently supported by
electrophysiological studies that found different types of Cl±
channels with different conductance on the sperm head (Bai and
Shi, 2001). A Cl± ef¯ux is feasible for inducing a depolarization of
the RP since it has been shown that sperm contain a high amount of
Cl± ions internally (Sato et al., 2000). Moreover, a Cl± ef¯ux seems
to be induced by progesterone (Meizel, 1997).
Although the role of Ca2+ in inducing the AR is well
demonstrated, and Ca2+ elevation has been shown to be mediated
by ion channels, the mechanism by which the ZP or other agents
are able to gate the channels is not yet understood. Figure 2
(bottom) demonstrates a schematic representation of the sperm
plasma membrane ion channel activity during the processes of
chemotaxis and AR.
Downloaded from http://humupd.oxfordjournals.org/ at Pennsylvania State University on February 23, 2013
Figure 2. A schematic representation of ion channel activity. (Top) Bovine oocyte plasma membrane during maturation and fertilization processes (from Tosti
et al., 2000, 2002; Boni et al., 2002). At the germinal vesicle stage (left), there is a preponderance of K+ and L-type Ca2+ channels, which decreases
signi®cantly at the metaphase II stage (middle). At fertilization, the spermatozoon induces a release of calcium from the intracellular stores that gates the Ca2+activated K+ channels. Simultaneously, plasma membrane Ca2+ channels allow calcium entry through the plasma membrane. (Bottom) Animals and human
sperm plasma membrane during chemotaxis and acrosome reaction (AR) processes (from Darszon et al., 1999; 2001, 2002). Main channels playing a role
during the two processes are reported. In and out represent intracellular and extracellular environments.
Ion channels in gametes
Electrical events at fertilization
Figure 3. The depolarization step (arrow), observed by De Felice and
colleagues at the Zoological Station in Naples in 1978 on sea urchin eggs at
the moment of fertilization, represents the ®rst electrical response of the
oocyte to a single spermatozoon interaction ever recorded, preceding the
fertilization potential by >10 s. Reprinted by permission from Nature (Dale
et al., 1978), ã 1978 Macmillan Publishers Ltd.
mechanism for the regenerative Ca2+ waves during fertilization
(Eisen et al., 1984; Swann and Whitaker, 1986).
Electrical changes of the oocyte plasma membrane represent the
other crucial event of oocyte activation, along with the rise in
intracellular Ca2+. The relationship between these two events
varies among species, and is not yet clear. Hagiwara and Jaffe
(1979) have demonstrated that an oocyte is not activated by
electrical modi®cations alone, and that intracellular calcium
modi®cations are not strictly dependent on electrical changes.
However, these two events occur almost simultaneously, with
similar dynamics, and they are linked by common mechanisms. A
clear example of the linkage between these two events is given by
calcium-activated K+ channels in mammalian oocyte activation. In
other species, the linkage between electrical changes and calcium
rise varies from a strict dependence as in annelids (Eckberg and
Miller, 1995) to completely separate pathways as in ascidian (Dale,
1987).
A role for K+ ion ¯uxes through the plasma membrane in the
process of oocyte activation was ®rst described in pioneering
studies on marine animals (Tyler et al., 1956; Hiramoto, 1958).
The spermatozoon generates a transient change of the RP in the
oocyte, described as the fertilization potential (FP). However, the
biophysical origin of this voltage modi®cation was not elucidated.
Using the voltage-clamp technique, it was later demonstrated that
the FP results from an ion ¯ux (the fertilization current: FC) across
the plasma membrane. Further characterization demonstrated that
this current is due to the gating of novel plasma membrane ion
channels in the newly fertilized oocyte (for reviews see Miyazaki,
1988; Dale and De Felice, 1984, 1990). Pharmacological agents
generated similar ion activation currents in oocytes mimicking the
form and the effects of the sperm-induced ion current, resulting in
the early development of parthenogenetic embryos (Boni et al.,
2002; Tosti et al., 2002).
During the 1950s, electrical parameters and ion contents were
shown to change at fertilization in echinoderm oocytes (MonroyOddo and Esposito, 1951; Hiramoto, 1959). In star®sh oocytes, a
FP is the ®rst electrical event of fertilization (Dale et al., 1981),
and an inward ion current underlies the FP (Lansman, 1983). In sea
urchin oocytes, Dale et al. (1978) characterized a typical FP as a
59
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Fertilization is a highly specialized process of cell±cell interaction
that marks the creation of a new and unique individual. It is a
complex multi-step process involving many events, including
gamete recognition, binding and fusion. Reciprocal activation of
the two gametes is a crucial element of these events; signals from
the oocyte investments induce dramatic changes in form and
function of the spermatozoon, and the spermatozoon triggers the
quiescent oocyte into metabolic activation (for review see
Yanagimachi, 1994). Oocyte activation is a dynamic mechanism,
and its progress is characterized by early and late events (Xu et al.,
1994). Cortical granule exocytosis is an early activation event, and
resumption of meiosis is a late activation event.
The ®rst described metabolic ®ndings associated with oocyte
activation were described in the sea urchin, an increase in O2
consumption and activation of NAD kinase (Epel, 1978). At
fertilization, an increase in internal pH occurs in sea urchin oocytes
is required for the initiation of development (Johnson et al., 1976).
This is not the case for mammalian oocytes, where no detectable
change in pH occurs after fertilization (Baltz et al., 1995).
However, the ®rst well-recognized event driving oocyte
activation is a sperm-induced Ca2+ release (for review see
Swann and Jones, 2002). An increase in intracellular Ca2+ at
fertilization was observed for the ®rst time in the oocytes of the
medaka ®sh (Ridgway et al., 1977). This mechanism was
subsequently universally recognized, in both animal and vegetable
kingdoms (Roberts et al., 1994; Digonnet et al., 1997). Ca2+ is
therefore considered to be the major second messenger during
oocyte activation (Whittingham, 1980; Whitaker and Steinhardt,
1982; Jaffe et al., 1983; Kline, 1988; Kline and Kline, 1992;
Swann and Ozil, 1994; Lawrence et al., 1997). However, the
mechanism by which sperm can evoke this event is not yet
characterized. Three theories are currently under investigation,
named by Jaffe (1991) as `conduit, contact and content' models
(for review see Nixon et al., 2000). In the `conduit' model the
sperm acts as a conduit for Ca2+ entry from the extracellular
medium (Creton and Jaffe, 1995). The `contact' model assumes
the activation of an oocyte phospholipase C following sperm±
oocyte interaction (Foltz and Shilling, 1993; Evans and Kopf,
1998). The `content' model proposes that a soluble sperm factor is
released into the oocyte following gamete fusion (Dale et al.,
1985; Swann, 1990). Regardless of the mechanism, it is certain
that sperm acts as a preliminary stimulus for oocyte activation. The
activation signal from the site of sperm entry is transmitted to the
entire oocyte, mediated by an increase in intracellular Ca2+ that
quickly returns to baseline. This calcium wave represents a single
event in some species, such as medaka ®sh (Ridgway et al., 1977),
sea urchin (Steinhardt et al., 1977) and frog (Busa and Nuccitelli,
1985). In other species, such as mammals and ascidians, the wave
is followed by a long-lasting series of Ca2+ oscillations (Miyazaki
and Igusa, 1981b; Cuthbertson and Cobbold, 1985). The mechanism of propagation, ampli®cation and regeneration of this Ca2+
signal is mediated by Ca2+-speci®c receptors (Fujiwara et al.,
1990; Carroll and Swann, 1992; Swann, 1992; Whitaker and
Swann, 1993; Miyazaki et al., 1993; Yue et al., 1995; Galione
et al., 2000). Among these, ryanodine receptors are considered to
be involved in Ca2+-induced Ca2+ release (CICR), a plausible
E.Tosti and R.Boni
Table I. Type of ion channels involved in oocyte and sperm activation and membrane potential modi®cations at oocyte activation/fertilization
Species
Ion channels involved in:
Oocyte activation
Sea urchin
Non-speci®c, Na+, Ca2+
Ascidians
Amphibians
Mouse
Non-speci®c
Cl±, K+, Na+; Ca2+activated Cl±
Ca2+-activated K+
Hamster
Bovine
Human
Ca2+-activated K+
Ca2+-activated K+
Ca2+-activated K+
Sperm activation (AR)
Na+, H+, Ca2+, K+
T-type Ca2+
References
Depolarization, +17 mV
Dale et al., 1978; De Simone et al., 1998
GonzaÁlez-MartõÁnez et al., 1992
Dale et al., 1983; Dale and De Felice, 1984
Webb and Nuccitelli, 1985a,b;
Glahn and Nuccitelli, 2003
Igusa et al., 1983
Arnoult et al., 1996
Miyazaki and Igusa, 1981b
Tosti et al., 2002
Gianaroli et al., 1994; Dale et al., 1996
Garcia and Meizel, 1999; Sato et al., 2000;
Bai and Shi, 2001; Rossato et al., 2001
Depolarization, +60 mV
Depolarization, +22 mV
Hyperpolarization, ±22 mV
Hyperpolarization, ±42 mV
Hyperpolarization, ±32 mV
Hyperpolarization, ±40 mV
AR = acrosome reaction; RP = resting potential.
depolarization, preceded by a depolarizing-like step; these events
were accompanied by an increase in voltage noise and a decrease
in membrane resistance (Figure 3). The FP form was attributed to
the activation of a transient voltage-dependent inward current, due
to the increase in intracellular Ca2+ concentration occurring at
fertilization (David et al., 1988). Initial characterization of ion
channels responsible for the FC demonstrated that non-speci®c ion
70 pS single channel conductance (Dale et al., 1978) and voltagegated Na+ and Ca2+ channels (Chambers, 1989b) are involved.
More recently, De Simone et al. (1998) recorded an inward Ca2+dependent FC in whole cell voltage-clamped sea urchin oocytes,
demonstrating that this current is driven by non-speci®c ion
channels.
In the oocytes of Ciona intestinalis, the spermatozoon induces
an inward FC via a new population of channels that appears in the
membrane a few seconds after insemination (Dale and De Felice,
1984). Gating of these channels is accompanied by a small steplike depolarization followed by a larger overshooting depolarization (Dale et al., 1983). These fertilization channels have been
characterized as large and non-speci®c, with a single conductance
of 400 pS (Dale and De Felice, 1984; De Felice and Kell, 1987).
Recently, Tosti et al. (2003) have demonstrated a role for FC in
triggering normal embryo development in C. intestinalis. A
different pattern of voltage-gated channels was described in
Phallusia mammillata after fertilization, where voltage-gated Ca2+
channels play a role (Goudeau and Goudeau, 1993) and an inward
rectifying Cl± current appears after fertilization in Boltenia villosa
(Coombs et al., 1992). A summary of RP modi®cations and the
major channels involved in oocyte activation and fertilization are
reported in Table I.
Oocyte Cl± channels are responsible for the FP in several
amphibian species. In Xenopus laevis, a membrane depolarization
at fertilization is in¯uenced by the external Cl± concentration
(Webb and Nuccitelli, 1985a,b); this is a consequence of Cl± ion
ef¯ux (Cross and Elinson, 1980). Similarly, a positive shift in FP
was shown in R. pipiens associated with an increase of either K+ or
Cl± and a decrease in Na+ conductance (Jaffe and Schlichter, 1985;
Schlichter, 1989b). Membrane potential changes in R. cameranoi
oocytes are also based on K+ as well as on Cl± conductance
60
(Erdogan et al., 1996), with a different function for these two ions.
Cl± ions are apparently responsible for the ®rst depolarization
phase evoked by sperm, whereas K+ contributes to the repolarizing
phase. The role of Ca2+ channels in amphibian FP is less clear.
Although no signi®cant role for external Ca2+ has been shown in R.
cameranoi (Erdogan et al., 1996), there is clear evidence that
intracellular Ca2+ contributes to gating the Cl± channels (Barish,
1983). Recently, Glahn and Nuccitelli (2003) recorded the FC in
Xenopus oocytes for the ®rst time; this FC resembles that of
marine invertebrates, but it is generated by Ca2+-activated Cl±
channels. Studies in hamster oocyte demonstrated a series of
hyperpolarizations following fertilization, the ®rst marked difference shown between mammalian and non-mammalian electrical
responses (Miyazaki and Igusa, 1981a). Similar FC have subsequently been recorded in other mammalian species, such as mouse
(Igusa et al., 1983; Jaffe et al., 1983), human (Gianaroli et al.,
1994) and bovine (Tosti et al., 2002). The pattern in rabbit oocytes
is different, in that a preliminary depolarization is followed by a
repeated diphasic (hyperpolarization/depolarization) pattern of
membrane modi®cations (McCulloh et al., 1983). As in the marine
models, these electrical modi®cations of the plasma membrane
following fertilization in mammals are due to ion passage.
However, in contrast to the marine species, where FC is apparently
directly gated by the spermatozoon, in mammals Ca2+-activated
K+ channels are gated by an initial calcium release (Miyazaki and
Igusa, 1981b; Dale et al., 1996; Tosti et al., 2002). A dependence
between calcium modi®cation and plasma membrane potential
was argued by Miyazaki (1989) based on biophysical evidence
together with the fact that: (i) loading the oocyte with EGTA
prevents hyperpolarization oocyte and (ii) Ca2+ injection into the
oocyte triggers a similar hyperpolarization. The hyperpolarization
that follows fertilization is the ®rst of a series of electrical
oscillations of the membrane potential, in accordance with Ca2+
oscillations (for review see Miyazaki, 1989) that continue up to
pronuclear formation (Jones et al., 1995). These oscillations
decrease in frequency and amplitude during their progression. The
oscillations are large in hamster (Miyazaki and Igusa, 1983) and
smaller in mouse (Igusa et al., 1983; Jaffe et al., 1983) and rabbit
(McCulloh et al., 1983) oocytes. After oocyte activation, plasma
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T-type Ca2+, Ca2+activated, K+, Cl±
Change of RP at
oocyte activation
Ion channels in gametes
Concluding remarks
The presence of ion currents in gamete plasma membranes and
their modi®cation during the maturation and fertilization processes
are described from marine invertebrates to humans. Nonetheless,
their role in gamete biology is not completely understood. For
many years the most feasible hypothesis has been that the
electrical change in oocytes at fertilization was devoted to a rapid
block to polyspermy. However, recent studies reported in this
review shed new light on the role of ion currents in many processes
of gamete biology. A tremendous potential of knowledge is now
emerging from studies on ion channels in sperm physiology. The
molecular cloning of an ion channel speci®c for mouse and human
testis and sperm (Quill et al., 2001; Ren et al., 2001) has clearly
shown that sperm ion currents are involved in male fertility.
Ion channels can be used to elucidate ion current signalling
patterns, and may provide new potential tools to help clarify the
mechanism of fertilization and to improve both assisted reproductive and contraceptive technologies.
Acknowledgements
We thank Dr K.Elder and Dr L.J.De Felice for their helpful comments
and Mr G.Gargiulo for ®gure preparation. This work was supported by
the Italian Ministry of University and Research (M.I.U.R.), COFIN
2002 project.
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