of ions and ion channels in capacitation and acrosome reaction of spermatozoa
B Purohit, Malini Laloraya, G. Pradeep Kumar
of Life Sciences, Devi Ahilya University, Vigyan Bhavan, Khandwa Road,
Indore 452001, M.P., India.
Asian J Androl 1999 Sep; 1: 95-107
AbstractCapacitation and acrosome reaction are important prerequisites of the fertilization process. Capacitation is a highly complex phenomenon occurring in the female genital tract, rendering the spermatozoa capable of binding and fusion with the oocyte. During capacitation various biochemical and biophysical changes occur in the spermatozoa and the spermatozoal membranes. Ions and ion channels also play important roles in governing the process of capacitation by changing the fluxes of different ions which in turn controls various characteristics of capacitated spermatozoa. Along with the mobilization of ions the generation of free radicals and efflux of cholesterol also plays an important role in the capacitation state of the spermatozoa. The generation of free radical and efflux of cholesterol change the mechano-dynamic properties of the membrane by oxidation of the polyunsaturated lipids and by generating the cholesterol free patches. The process of capacitation renders the spermatozoa responsive to the inducers of the acrosome reaction. The glycoprotein zona pellucida 3 (ZP3) of the egg coat zona pellucida is the potent physiological stimulator of the acrosome reaction; progesterone, a major component of the follicular fluid, is also an inducer of the acrosome reaction. The inducers of the acrosome reaction cause the activation of the various ion-channels leading to high influxes of calcium, sodium and bicarbonate. The efflux of cholesterol during the process of capacitation alters the permeability of the membrane to the ions and generate areas which are prone to fusion and vesiculation process during the acrosome reaction. This review focuses mainly on effects of the ion and ion-channels, free radicals, and membrane fluidity changes during the process of capacitation and acrosome reaction.
The independent discovery by Austin (1951) and Chang (1951) that the spermatozoa of eutherian mammals needs a few hours residence time in the female reproductive tract before acquiring the capacity for fertilization, has provided one of the most enduring puzzles of reproductive biology. The process was earlier termed as capacitation. Capacitation can occur in vitro spontaneously in a defined medium without the addition of biological fluids, which suggests that this process is intrinsically modulated by the spermatozoa itself such that these cells are preprogrammed to undergo capacitation when they are incubated in the appropriate medium. Our knowledge regarding this phenomenon has been derived from in vitro studies.
Although numerous hypotheses have been developed, the precise nature of capacitation is still obscure. Changes associated with sperm capacitation include an increase in respiration and subsequent changes in the motility pattern, called as hyper-activation which is characterized by pronounced flagellar movements and a marked lateral excursion of sperm head in a non-linear trajectory, and in a number of species, removal of cholesterol from the sperm plasma membrane, destabilization of sperm membranes, an increase in intracellular pH and calcium levels, activation of second messenger systems[8-10], and/or removal of zinc. The most important change in sperm after capacitation is its ability to undergo acrosome reaction (AR) in response to ZP3, progesterone[4,12] and calcium ionophores. The responsiveness of spermatozoa to ZP3[4,14] and progesterone[15-18] increases during capacitation. Capacitation is also associated with changes in sperm plasma membrane fluidity, intracellular changes in ionic concentration, and sperm cell metabolism. Capacitation in sperm does not occur synchronously and is a transient and irreversible process. Various reviews have been published till date on the process of capacitation[4,21-23]. This review focuses on the changes occurring in the sperm plasma membrane during capacitation, leading to the changes on the surface of sperm membrane, flow of electrolytes and modification of membrane during/after capacitation.
Role of ion channels in capacitation
environment and ionic fluxes through membrane are highly important in
the spermatozoal maturation, capacitation and in initiating the process
of gamete interaction. The various types of ion channels are observed
in the mammalian sperm plasma
membrane, suggesting their range of different roles in sperm physiology
and gamete interaction.
Mammalian spermatozoa possess several [Ca2+]i regulatory systems, including a pathway that is similar to the L-type of voltage-sensitive Ca2+ channel[25-28] that has been characterized in a variety of somatic cell types[29,30]. Two types of agonist-dependent [Ca2+]i transport pathways are defined. The first mediates small transient [Ca2+]i elevations that are restricted to the sperm head. Dye emission and quenching studies ineicate that this focal channel is not voltage-regulated, conducts several divalent metal cations (Co2+, Mn2+ & Ni2+) in addition to Ca2+ and has properties of a poorly selective cation channel. The second transporter mediates sustained [Ca2+]i elevation through out the cells and is pharmacologically identical to the L-type of voltage sensitive calcium channel. These channels are distinguished by inhibitor sensitivity and by regulation during sperm maturation.
major candidates have been identified in mammalian sperm cells for modulation
of [Ca2+]i. ( i ) a Ca2+-ATPase capable
of pumping Ca2+ out of the cell; (ii) a Na+/Ca2+
exchanger proposed to effect a [Ca2+]in/[Na+]out
exchange, and (iii) Ca2+ channels
that would permit a rapid Ca2+ influx[31,32]. Of
the three Ca2+ modulating mechanisms known to exist in mammalian
plays a major role during the process of capacitation as indicated by
published reports favoring its presence on sperm head of mouse[33,34],
bull, human as observed by changes in chlortetracycline
pattern. Since most somatic cell Ca2+-ATPases are calmodulin-sensitive,
it is possible that the sperm may possess the same
enzyme as well. Relatively short incubation in presence of a calmodulin
inhibitor trifluoroperazine or napthalensulfonamide (W-7) accelerated
the capacitation of bull, human, and mouse spermatozoa[34-36,38].
presence of Na+/Ca2+ exchanger is documented on
the sperm, its role in controlling the intracellular Ca2+ during
capacitation is not clear. A low molecular weight protein, caltrin initially
reported to be present in the bovine seminal plasma[39,40].
It has been shown to be present on ejaculated bovine sperm but
not on the membranes of epididymal sperm. Caltrin was also
reported to be present
on the male reproductive tracts of guinea pigs, mouse and
rats, which inhibits the Na+/Ca2+
exchanger to maintain the intracellular Ca2+ at low levels.
In the female genital tract, conformational changes abolished the
activity of caltrin protein and stimulated the exchange to induce a [Ca2+]in/[Na+]out movement.
The presence of Ca2+ channels is heavily documented in
mammalian spermatozoa but their role in modulating sperm intracellular
calcium during capacitation is still controversial.
calcium, intracellular levels of potassium (K+),
sodium (Na+) and
chloride (Cl-)[38,47] have been shown to be modulated
during capacitation. Monovalent
cationic ionophores monensin and nigericin stimulated rapid acrosome reaction
in guinea pig spermatozoa in presence of extracellular sodium, calcium
and bicarbonate (HCO3/CO2). It has been suggested
that ionophore induced rise in the intracellular Na+ concentrations
in the sperm is a pre-Ca2+ entry event, that stimulates endogenous
Ca2+/Na+ exchanger[48,49] which is important
for capacitation. K+ is shown to be non-essential for capacitation,
but is essential for
acrosome reaction of mammalian spermatozoa. Spermatozoa are shown to be
capacitated in K+ free or K+ deficient media.
Monovalent ions like K+, Rb+, Cs+ can
replace Na+ at a high concentration, whereas at lower concentrations
they are ineffective. Recently a pH-dependent K+-
channel had been identified and cloned and its dependence on pH and membrane
An increase in the intracellular pH (pHi) has been observed during capacitation[10,52,53]. This increase in pH is attributed to the bicarbonate anion. The requirement of this anion has been well established in mouse[54-57], hamster and bull, although it remains to be demonstrated in other mammalian species. Bicarbonate acts through stimulation of c-AMP metabolism, since the mammalian sperm adenylyl cyclase is stimulated by bicarbonate[60-62]. It is interesting that bicarbonate levels are low in epididymis and high in seminal plasma and oviduct. The presence of bicarbonate in the extracellular milieu has been positively correlated with the motility of sperm. Changes in the concentration of bicarbonate in the male and female reproductive tracts could play an important role in the suppression of capacitation in the epididymis and the promotion of this process in vivo in the female reproductive tract.
is present in high concentration in seminal fluid and spermatozoa compared
to blood plasma. Numerous studies indicate that elevated
zinc levels are responsible for maintaining sperm in a quiescent state
and for stabilizing sperm membranes during epididymal storage,
ejaculation, modulating acrosome reaction,
sperm motility and sperm chromatin decondensation.
The intracellular levels of zinc decrease in the acrosome of hamster spermatozoa during
capacitation and incubation of spermatozoa with zinc during capacitation inhibits
capacitation, once capacitated zinc has no effect in interfering with
acrosome reaction. These results indicate that zinc plays
an important role in destablizing plasma membrane during acrosome reaction.
Reactive oxygen species during mammalian sperm capacitation
oxygen species (ROS) are a group of oxygen free radicals consisting of superoxide
anion radical (O2),
singlet oxygen (O), hydrogen peroxide (H2O2)
and other organic peroxides. Of these superoxide radical,
hydrogen peroxide and
nitric oxide are of particular importance in the mammalian sperm physiology.
concentration og ROS are shown to cause sperm pathology like ATP depletion, leading
to insufficient axonemal phosphorylations, lipid peroxidation and loss
of motility and viability. The detrimental effects of ROS
are due to the peroxidative damage to the sperm plasma membrane[73-76].
Mammalian spermatozoa are susceptible to such damage because of their
high content of polyunsaturated fatty acids and relatively
low levels of antioxidants enzymes[78-81].
major source of ROS, that damage the spermatozoa are thought to be infiltering
white blood cells and the spermatozoa themselves[81,83,84].
It has been proposed that superoxide production by human spermatozoa is
dependent on the activity of a membrane bound NADPH oxidase regulated
by calcium and protein phosphorylation, which in turn is dependent on
protein kinase C[83,85], whereas the production of hydrogen
peroxide is accounted for by the activity of superoxide dismutase.
anion radical (O2),
plays an important role during maturation of spermatozoa
and in the control of sperm function through the redox regulation of
tyrosine phosphorylation. Mammalian spermatozoa are highly
sensitive to oxidative stress and defective sperm functions have been
associated with lipid peroxidation (LPO)[80,88-90]. At low
concentrations ROS are involved in the activation of certain enzymes[91-93].
Excess ROS generation is associated with a decrease in the fusion
rates between sperm and oocytes and inhibits the AR, motility
and fertilizing potential of spermatozoa[94,95]. O2
is shown to promote the capacitation of human sperm
and there is a superoxide radical surge in
the capacitated spermatozoa during the process. It is reported
that ( i ) exogenously
through xanthine/xanthine oxidase system induced hyperactivation and capacitation,
(ii) capacitating sperm produced elevated concentrations of O2
over prolonged periods of time and (iii) removal of this ROS by superoxide
dismutase (SOD) prevented
hyperactivation and capacitation. All these observations stress on the
importance of O2
in the process of capacitation and fertilization[98,99].
Along with O2,
hydrogen peroxide (H2O2) was also shown to
promote capacitation of human sperm. The mechanisms and
targets of action
of hydrogen peroxide are still unknown. However, it is implicated that
hydrogen peroxide acts on the sperm membrane surface[79,81].
Controlled oxidation of membrane surface thiol groups is also suggested
as one of the possible route through which hydrogen peroxide acts.
Another possible mechanism by which hydrogen peroxide could act is by
causing a decrease in diffusibility of lipids observed with the plasma
membrane of spermatozoa during capacitation, in order to creat fusion
areas for acrosome reaction.
oxide (NO) is a free radical synthesized in vivo during the conversion
of L-arginine to L-citrulline
by the enzyme nitric oxide synthases (NOS). It is reported to be the most
important messenger employed in a host of biological process.
High concentrations (1-100 mol/L) of the NO-releasing agents are shown
to bring about decrease in sperm motility, whereas lower
concentration of the
same product (50-100 nmol/L) improved the motility and viability of the
cryopreservation. Although no detectable amount of NOS
was observed on the
sperm, very high concentrations (5-20 mmol/L) of NOS inhibitors
were shown to reduce hamster sperm hyperactivation. Nitric
oxide did not promote capacitation by inducing the production of superoxide
from the sperm cells, indicating that NO promotes capacitation
by other mechanism(s), by stimulating one of the later steps of capacitation,
by passing the induction of superoxide production and sperm hyperactivation.
Catalase blocks capacitation induced by NONOates implying that hydrogen
peroxide is needed for the action of nitric oxide. There
are two pathways by which nitric oxide interacts with hydrogen peroxide.
First, NO can directly react with hydrogen peroxide to form singlet oxygen[109,110],
which is sufficiently reactive to cause oxidation of membrane lipids and thiol
groups. Second, nitric oxide can be further oxidized to
nitrosonium cation which then reacts with hydrogen peroxide to give per-oxynitrite
anion[109,110]. This anion is highly reactive and can act directly
on cellular compounds such as lipids or thiol containing molecules
or decompose to two other reactive species, nitrogen dioxide and hydroxyl
nitric oxide synthase enzyme is absent or is present at a very low levels
in the spermatozoa,
suggesting that the NO required for the capacitation in vivo are
generated or secreted in the female reproductive tract. Nitric oxide synthase is
reported to be present in the female reproductive tract[106,111].
These combined results indicates that capacitation is a part of an oxidative
stress and these three reactive oxygen species are involved in vivo in
sperm capacitation; the superoxide anion produced by the spermatozoa is
dismutated into hydrogen peroxide. The nitric oxide produced in the female
genital tract interacts with each other causing the controlled oxidation
of membrane components during the capacitation process.
Role of phospholipid and membrane fluidity
spermatozoa contain a very high concentration of polyunsaturated fatty acids
in the sperm membranes[112-114]. These fatty acids are required
to give the plasma membrane the fluidity needed to sustain important biochemical
and biological functions, including the maintenance of various membrane
bound enzyme activity and completion of membrane fusion events associated
with acrosome reaction and union with oocyte. The membrane fluidity is
maintained by the controlled peroxidation of the membrane phospholipids
by reactive oxygen species[115-117]. Membrane fluidity is reported
to play an important role in sperm maturation in mice
and ram. Changes in the sperm plasma membrane lipids and
phospholipids are yet another important phenomenon. These changes include
the increase in the
membrane fluidity[4,102], increase in the membrane potential/polarity during
the process of mammalian sperm capacitation. The increased levels of superoxide
radical during the process of capacitation leads to the increase in the
membrane fluidity of the sperm membranes by modifying the
local repulsive strain and hydration barrier, which leads to the vesiculation
of the membranes during acrosome reaction.
The other change which takes place during capacitation is the removal of cholesterol from sperm plasma membrane. Cholesterol is known to regulate the fluidity of the membrane lipid bilayers and the permeability of membrane and to modulate the lateral mobility of integral proteins and functional receptors within the membrane[121,122]. Cholesterol/phospholipid (C/PL) ratio of the sperm determines the capacitation state of the sperm[123,124]. The actual moiety that stabilizes the sperm membrane is cholesterol sulfate and it increases 18-fold during the epididymal transit of sperm in hamster. A freshly ejaculated sperm has a high C/PL ratio; and during capacitation this ratio falls. The red blood cell has a C/PL molar ratio of 0.9 and the C/PL molar ratios of the mammalian sperm ranges from 0.20-0.80. These data argue against the view that a recently ejaculated sperm is incapable of acrosomal exocytosis because its plasma membrane is somehow frozen by an extremely high concentration of cholesterol.
has been postulated that cholesterol sulfate regulates the fluidity of
the sperm membrane during epididymal maturation, capacitation and acrosome
reaction. The action of the cumulus sulfatases on the
cholesterol sulfate is one part of the mechanism of the capacitation.
There are cholesterol rich and cholesterol deficient regions on the head
of the spermatozoa and during in vitro capacitation loss of cholesterol
takes place to form the cholesterol-free patches and that
these patches are the sites of fusion at the time of AR.
The removal of this sterol could account for the increase in membrane
fluidity which would allow greater lateral movements of integral membrane
proteins, and a greater permeability to calcium that occurs during capacitation,
which are key triggers for the acrosome reaction. Bovine
serum albumin (BSA), which is present in
the capacitation media for mammalian sperm, is believed to function as
a sink for the removal of cholesterol from the sperm plasma membrane[123,128,130,133,134].
However, it is implicated that bovine serum albumin along with bicarbonate
causes the destabilization of the membrane during the capacitation process
but not the BSA alone.
2 Acrosome reaction (AR)
mammalian acrosome, a cap-like membrane limited organelle which covers
the anterior part of the nucleus on the sperm head, has been described
as a secretory granule.
The AR in mammals involves the fusion, vesiculation (fenestration) and
loss of outer acrosomal membrane and its overlying sperm plasma membrane
and the release of acrosomal matrix material. Acrosome
reaction is an exocytic event characterized by fusion of the outer acrosomal
membrane and the sperm plasma membrane allowing the release of the acrosomal
contents. During this process hybrid membrane vesicles are formed, giving
rise to a patchy pattern seen when sperm are stained with FITC labeled
lectins. This organized membrane fusion and vesiculation
is required for sperm penetration through the acellular coating enclosing
the egg. This exocytic process involves the anterior region
of head and is not
extended beyond the equatorial segment. Spontaneously AR
occurs at a very low level, which occurs due to the self
aggregation of sperm
receptor for zona pellucida. Another hypothesis is that
the Na+ and/or
Ca2+ pumping mechanism becomes less efficient with time, which
results in a gradual increase in intracellular Ca2+ and pH,
leading to spontaneous AR.
physiological and in vitro conditions, the egg specific extracellular
matrix, the zona pellucida, stimulates acrosomal exocytosis in mammalian
sperm[4,27,138]. One of the zona pellucida glycoprotein ZP3,
which is a sulphated glycoprotein,
stimulates AR[4,139,140]. Progesterone and its analogue 17-OH-progesterone,
a major component of follicular fluid, had been found to rapidly induce
AR in mammalian sperm[12,141-145]. AR can be induced in vitro
by ionophores which exchange Ca2+ for other ions such as H+
aggregation is the first event that occurs in spermatozoa stimulated
by ZP3 and progesterone[146-148].
This receptor aggregation is followed by a cascade of membrane and cytosolic
changes involved in the mammalian sperm AR. The contributing role of ions
and ion channels and membrane factors during the AR is discussed.
Role of ions and ion channels during the mammalian sperm acrosome reaction
of the agents responsible for the initiation of the AR is Ca2+.
Sea urchin and starfish spermatozoa do not undergo AR in response to egg
jelly substance when Ca2+ is deficient or absent from the medium.
The failure of fertilization of the eggs of the sea urchin and of other
marine species is due to specific inhibition of the AR.
Calcium plays an important role in the mammalian sperm acrosome reaction
as spermatozoa of all mammalian species do not initiate their acrosome
reaction in the absence of calcium[4,150-152].
of intracellular free calcium concentration in small cells became possible
using fluorescent calcium indicators, and it has been
measured in spermatozoa of various species[154,155]. Using
the intracellular calcium indicator, Quin-2, the mean resting level of
[Ca2+]i in human sperm was found to be approximately
150 nm and treating the sperm with ionomycin was shown to increase [Ca2+]i
ionophores are the most widely used nonphysiological inducers of AR.
AR can be induced in the absence of extracellular calcium with some agonists
in capacitated and non-capacitated spermatozoa[156-158]. Millimolar
levels of extracellular calcium is required for ZP3-induced acrosomal
the fluorescent probe Fura-2 the amount of [Ca2+]i
in response to addition of ZP3 was studied in mammalian spermatozoa[27,159].
Addition of ZP3 led to a rapid (2-5 min)
increase in the amount of [Ca2+]i, followed by a
plateau phase after 10-15 minutes[27,159]. Acrosome reaction
occurs during the sustained phase of calcium increase.
Activation of sperm L channels is required for ZP agonist-initiated exocytosis
and involves pertussis toxin sensitive GTP binding proteins[159,161].
The blocking of calcium channels by channel blockers inhibit the ZP -induced
a major component of human follicular fluid, initiates AR
by calcium influx[27,141,162,163]. During progesterone initiation
of the human sperm AR, there
is a several fold transient increase in [Ca2+]i,
after a few seconds of steroid addition[141,142,164]. This
rapid influx of calcium appears to be mediated
by a different set of calcium channels[18,165] as it is not
dependent on the pertussis toxin sensitive GTP-binding proteins and the
voltage sensitive calcium channels. Extracellular calcium is the absolute
requirement for the induction of acrosomal
exocytosis by progesterone. Published reports suggests
that calcium can be stored in the mammalian sperm[166,167].
Thapsigargin (50-500 mol/L),
a highly specific inhibitor of endoplasmic reticulum Ca2+-ATPase
Ca2+-pump, can initiate AR in capacitated sperm.
According to one hypothesis, thapsigargin induces
the release of calcium from the intracellular stores, which in turn leads
to a massive influx of extracellular calcium. In many
other cells, the endoplasmic reticulum is the site of such a Ca2+-store,
but there is no obvious endoplasmic reticulum in the cytoplasm of mature
sperm. Thapsigargin would not mobilize any mitochondrial stores. Hypothetical
calcium storage sites include the nucleus and the outer acrosomal membrane[166,169].
Interestingly, receptors for inositol trisphosphate (IP3), a physiological
releaser of intracellular calcium stores on the outer acrosomal membrane
of the rat sperm, may act as a calcium store. Calretuculin,
a calcium binding endoplasmic
reticulum protein involved
in calcium release, has been described in the rat acrosome.
may interact with the polar head groups of phospholipids, thus overcoming
the repulsion forces and allowing the approximation of the two membrane.
It is suggested that calcium may achieve this by causing condensation
of polar phospholipid head group, thus increasing hydrophobic attraction
forces between membranes by exposure of excess of hydrophobic groups in
the interior of the bilayers. The temporal and spatial
location of intracellular calcium granules was monitored during acrosome
reaction in ram spermatozoa. Calcium is initially associated with the
outer acrosomal membrane. As the process progresses, calcium associates
with the fusion sites between the outer acrosomal membrane and the plasma
membrane anterior to the equatorial segment. At later stages, calcium
is localized in both post acrosomal dense lamina and on outer acrosomal
membrane under the
equatorial segment. This finding suggest that calcium may be implicated
in the fusion process.
of other ions like sodium[34,176], chloride[177,178],
bicarbonate, and hydrogen[160,176] occur during
the AR, suggesting that besides calcium, other ions also play a role in
the process of calcium-dependent fusion of acrosomal membrane and the
sperm plasma membrane. Sodium is reportedly not required for the progesterone-initiated
human AR and the progesterone-mediated increase in intracellular calcium
is higher in the absence of sodium ions. It has been suggested
by the same workers that progesterone activates two channels, a calcium
and a sodium channel. However another report suggests
that in the absence of sodium ions, progesterone-mediated increase in
calcium and AR is inhibited. Chloride movement by the
mammalian sperm has been reported to occur during the zona- and progesterone-initiated
AR. Studies replacing bromide for chloride inhibits the zona initiated
AR. Wistrom and Meizel indicated that chloride
was required for the progesterone-initiated human sperm AR and that a
unique sperm progesterone receptor/chloride channel resembling
a neuronal gamma amino butyric acid A receptor/chloride channel
is present in these cells. Involvement of such a receptor/chloride channel
during the progesterone initiated AR in mouse has been well documented[145,180].
Involvement of glycine receptor/chloride channel along with GABA
receptor/chloride channel is also reported on the basis of inhibitor studies
on porcine and human spermatozoa. The role of bicarbonate
in the progesterone initiated human sperm AR has been reported.
Progesterone-initiated AR causes an increase in the cytosolic chloride
via the steroid receptor/chloride channel and would lead
to activation of the sperm bicarbonate/chloride exchanger, producing a
large bicarbonate influx and a chloride efflux. Bicarbonate is known to
stimulate AR by raising the pH and/or increasing the adenylate cyclase
activity, both of which appear to be important during the AR.
It has already been shown
that mammalian adenylate cyclase is stimulated by bicarbonate[61,62].
Bicarbonate/chloride exchanger has been located in the equatorial segment
region of the sperm
head. Bicarbonate plays an important role in regulating
the membrane fluidity of the sperm during the process of AR.
Bicarbonate along with BSA is shown to regulate the membrane fluidity,
but not the BSA alone.
Free radicals in acrosome reaction
of ROS in the mammalian sperm capacitation is very well documented, but
reports on their involvement in the acrosome reaction are very scanty.
Superoxide anion production is shown to be a part of ionophore induced
acrosome reaction[87,185]. Superoxide anion production drops
suddenly after the addition of the AR inducers, but is
highest during the capacitation process, indicating that it plays a major
part during the capacitation process rather at the time of acrosome reaction.
Hydrogen peroxide is known to induce hyperactivation and promote capacitation,
but is not involved in the acrosome reaction of the spermatozoa.
Membrane fluidity and acrosome reaction
anterior acrosome region of the human sperm plasma membrane, due to its
of antifusogenic sterols, seems to be resistant to immediate fusion. It
is the formation of sterol-depleted patches in the anterior acrosomal
region that renders it susceptible to membrane fusion.
Lipid distribution during capacitation appears to provide the fusogenic
domains required for membrane fusion in the acrosome reaction.
The most important consequences of the cholesterol efflux are the massive
influx of extracellular calcium, a prerequisite for the acrosome reaction.
The entrance of calcium may be due to the changes in the fluidity of the
membrane that renders the membrane permeable to calcium. This influx
opens the voltage-dependent calcium channels by stimulatory action of
calcium on phospholipase C.
Several calcium-dependent biochemical consequences occur, including
the activation of phospholipase C, activation of phospholipase
A2, activation of protein kinase C,
activation of enzymes of cAMP metabolism, leading to the
modification of phospholipid composition of membranes facilitating the
fusion events. Increased intracellular calcium can trigger different pathways
involved in the acrosome reaction: generation of diacylglycerol (DAG)
through phosphoinositide breakdown, DAG stimulation of phospholipase A2
and participation in the membrane fusion itself. Phospholipase A2
action on phospholipids gives rise to lysophospholipids and arachidonic
acid or other fatty acids, which are known to be highly fusogenic[191,192].
Calcium could act directly on negatively charged membrane lipids, by neutralizing
anionic phospholipids or cholesterol sulfate. This may induce membrane
destabilization and fusogenic intermediates formation during the acrosome
This work was supported by a CSIR grant 37(871)95-EMR-II to Kumar GP. Purohit SB received a Senior Research Fellowship ( 9/30(57)/95-EMR-I) and Laloraya M is the recipient of Senior Research Associateship from CSIR, New Delhi, India.
Austin CR. Observations on the penetration of the sperm into mammalian
egg. Aust J Sci Res 1951; 4: 581-96.