1 Capacitation

The independent discovery by Austin (1951)^[1] and Chang (1951)^[2] 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^[3].  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^[4]. 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^[4]. 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^[4], removal of cholesterol from the sperm plasma membrane^[5], destabilization of sperm membranes^[6], an increase in intracellular pH and calcium levels^[7], activation of second messenger systems^[8-10], and/or removal of zinc^[11]. 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^[13]. 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^[4]. Capacitation in sperm does not occur synchronously^[19] and is a transient and irreversible process^[20]. 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.

1.1 Role of ion channels in capacitation

Ionic 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^[24].

Mammalian spermatozoa possess several [Ca^2+]_i regulatory systems^[21], 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 [Ca^2+]_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^[14].

Three 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 spermatozoa,  Ca²⁺-ATPase 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^[35], human^[36] as observed by changes in chlortetracycline pattern. Since most somatic cell Ca²⁺-ATPases are calmodulin-sensitive^[37], 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].

The 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^[41]. Caltrin was also reported to be present on the male reproductive tracts of guinea pigs^[42], mouse and rats^[43], which inhibits the Na⁺/Ca²⁺ exchanger to maintain the intracellular Ca²⁺ at low levels^[44]. 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^[44]. The presence of Ca²⁺ channels is heavily documented in mammalian spermatozoa but their role in modulating sperm intracellular calcium during capacitation is still controversial.

Besides calcium, intracellular levels of potassium (K⁺)^[45], sodium (Na⁺)^[46]  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^[50]. Monovalent ions like K⁺, Rb⁺, Cs⁺ can replace Na⁺ at a high concentration, whereas at lower concentrations they are ineffective^[50]. Recently a pH-dependent K⁺- channel had been identified and cloned and its dependence on pH and membrane potential established^[51].

An increase in the intracellular pH (pH_i) 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^[58] and bull^[59], 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^[63]. The presence of bicarbonate in the extracellular milieu has been positively correlated with the motility of sperm^[61]. 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.

Zinc is present in high concentration in seminal fluid and spermatozoa compared to blood plasma^[64]. Numerous studies indicate that elevated zinc levels are responsible for maintaining sperm in a quiescent state^[65] and for stabilizing sperm membranes during epididymal storage^[66], ejaculation^[67], modulating acrosome reaction^[68], sperm motility^[69] and sperm chromatin decondensation^[70]. 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^[64]. These results indicate that zinc plays an important role in destablizing plasma membrane during acrosome reaction^[64].

1.2 Reactive oxygen species during mammalian sperm capacitation

Reactive 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^[71]. Of these superoxide radical, hydrogen peroxide and nitric oxide are of particular importance in the mammalian sperm physiology^[72].

High 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^[72]. 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^[77] and relatively low levels of antioxidants enzymes^[78-81].

The major source of ROS, that damage the spermatozoa are thought to be infiltering white blood cells^[82] 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^[81].

Superoxide anion radical (O₂),  plays an important role during maturation of spermatozoa^[86] and in the control of sperm function through the redox regulation of tyrosine phosphorylation^[87]. 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^[74] and inhibits the AR, motility and fertilizing potential of spermatozoa^[94,95]. O₂ is shown to promote the capacitation of human sperm^[96] and there is a superoxide radical surge in the capacitated spermatozoa during the process^[97]. It is reported that ( i ) exogenously generated O₂ 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^[100]. 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^[101]. 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^[102].

Nitric 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^[103]. High concentrations (1-100 mol/L) of the NO-releasing agents are shown to bring about decrease in sperm motility^[104], whereas lower concentration of the same product (50-100 nmol/L) improved the motility and viability of the spermatozoa after cryopreservation^[105]. Although no detectable amount of NOS was observed on the sperm^[106], very high concentrations (5-20 mmol/L) of NOS inhibitors were shown to reduce hamster sperm hyperactivation^[107]. Nitric oxide did not promote capacitation by inducing the production of superoxide from the sperm cells^[108], 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^[106]. 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^[71]. 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^[109] or decompose to two other reactive species, nitrogen dioxide and hydroxyl radical^[109].

The 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.

1.3 Role of phospholipid and membrane fluidity

Mammalian 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^[118] and ram^[119]. 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^[120] 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^[97] 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^[125] and it increases 18-fold during the epididymal transit of sperm in hamster^[126]. A freshly ejaculated sperm has a high C/PL ratio; and during capacitation this ratio falls^[23]. The red blood cell has a C/PL molar ratio of 0.9^[127] and the C/PL molar ratios of the mammalian sperm ranges from 0.20-0.80^[128]. 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. 

It has been postulated that cholesterol sulfate regulates the fluidity of the sperm membrane during epididymal maturation, capacitation and acrosome reaction^[129]. The action of the cumulus sulfatases on the cholesterol sulfate is one part of the mechanism of the capacitation^[130]. 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^[131]. 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^[132]. 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^[135].

2 Acrosome reaction (AR)

The 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^[136]. 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^[137]. 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^[94]. This organized membrane fusion and vesiculation is required for sperm penetration through the acellular coating enclosing the egg^[137]. This exocytic process involves the anterior region of head and is not extended beyond the equatorial segment^[4]. Spontaneously AR occurs at a very low level^[4], which occurs due to the self aggregation of sperm receptor for zona pellucida^[138]. 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^[4].

Under 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⁺ and Na^+[13].

Receptor 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.

2.1 Role of ions and ion channels during the mammalian sperm acrosome reaction

One 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^[149]. 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].

Measurement of intracellular free calcium concentration in small cells became possible using fluorescent calcium indicators^[153], 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 significantly^[154].

Calcium ionophores are the most widely used nonphysiological inducers of AR^[4]. 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 exocytosis^[34], using 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^[160]. Activation of sperm L channels is required for ZP agonist-initiated exocytosis^[27] and involves pertussis toxin sensitive GTP binding proteins^[159,161]. The blocking of calcium channels by channel blockers inhibit the ZP -induced acrosome reaction^[161].

Progesterone, 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^[18]. 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^[168], can initiate AR in capacitated sperm^[169]. 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^[170]. 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^[171]. Calretuculin, a calcium binding  endoplasmic reticulum protein involved in calcium release, has been described in the rat acrosome^[172].

Calcium may interact with the polar head groups of phospholipids, thus overcoming the repulsion forces and allowing the approximation of the two membrane^[173]. 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^[174]. 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^[175].

Fluxes of other ions like sodium^[34,176], chloride^[177,178], bicarbonate^[179], 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^[176]. It has been suggested by the same workers that progesterone activates two channels, a calcium and a sodium channel^[176]. However another report suggests that in the absence of sodium ions, progesterone-mediated increase in calcium and AR is inhibited^[162]. 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^[47]. Wistrom and Meizel^[177] 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^[181]. The role of bicarbonate in the progesterone initiated human sperm AR has been reported^[179]. Progesterone-initiated AR causes an increase in the cytosolic chloride via the steroid receptor/chloride channel^[177] 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^[182]. 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^[183]. Bicarbonate plays an important role in regulating the membrane fluidity of the sperm during the process of AR^[184]. Bicarbonate along with BSA is shown to regulate the membrane fluidity, but not the BSA alone^[135].

2.2 Free radicals in acrosome reaction

Role 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^[97], 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^[100].

2.3 Membrane fluidity and acrosome reaction

The anterior acrosome region of the human sperm plasma membrane, due to its high concentration 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^[131]. Lipid distribution during capacitation appears to provide the fusogenic domains required for membrane fusion in the acrosome reaction^[186]. 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^[138]. Several calcium-dependent biochemical consequences occur, including the activation of phospholipase C^[187], activation of phospholipase A₂^[188], activation of protein kinase C^[189], activation of enzymes of cAMP metabolism^[190], 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 reaction^[193].

3 Conclusions

4 Acknowledgments

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.

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