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CAVEAT LECTOR
Elisabetta Baldi, Michaela Luconi, Lorella Bonaccorsi, Csilla Krausz and Gianni Forti.
Dipartimento di Fisiopatologia Clinica, Unita' di Andrologia, Universita' di Firenze, viale Pieraccini 6, I-50139 Firenze, Italy.
Received 05/30/96; Accepted 07/30/96; On-line 08/15/96
TABLE OF CONTENTS
Two processes, namely capacitation and acrosome reaction, are of fundamental importance in the fertilization of oocyte by spermatozoon. Physiologically occurring in the female genital tract, capacitation is a complex process, which renders the sperm cell capable for specific interaction with the oocyte. During capacitation, modification of membrane characteristics, enzyme activity and motility property of spermatozoa render these cells responsive to stimuli that induce acrosome reaction prior to fertilization. Physiological acrosome reaction occurs upon interaction of the spermatozoon with the zona pellucida protein ZP3. This is followed by liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona and exposes molecules on the sperm equatorial segment that allows fusion of sperm membrane with the oolemma. The molecular mechanisms and the signal transduction pathways mediating the processes of capacitation and acrosome reaction are only partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodelling in sperm plasma membrane as well as changes in protein phosphorylation.
The human and mouse sperm receptor for ZP3 has been recently sequenced and cloned. This receptor exhibits sequence homology with proto-oncogenes that mediate proliferation and differentiation in somatic cells. This review summarizes the main signal transduction pathways involved in capacitation and acrosome reaction.
2. SPERM CAPACITATION: BACKGROUND
The process of capacitation consists of a series of functional biochemical and biophysical modifications that render the ejaculated spermatozoa competent for fertilization of the oocyte. These fundamental processes normally take place in the female genital tract during the migration of spermatozoa to the site of fertilization (1). However, under appropriate conditions, capacitation can be also induced in vitro (1). Most of our knowledge regarding this process has been derived from in vitro studies. One of the functional consequence of capacitation is development of a distinct motility pattern called hyperactivation, which is characterized by pronounced flagellar movements, marked lateral excursion of the sperm head and a non linear trajectory. An additional manifestation of sperm capacitation is the acquisition of the ability to undergo acrosome reaction in response to physiological stimuli such as the zona pellucida protein ZP3 and progesterone (1). The responsiveness of spermatozoa to ZP3 (1-2) and to progesterone (3-5) increases during capacitation, assuring maximal responsiveness at the site of fertilization. Capacitation is associated with modifications in sperm surface protein distribution, alterations in plasma membrane characteristics, changes in enzymatic activities and modulation of expression of intracellular constituents (1). However the exact relationship among these modifications is not yet completely understood. In addition, there is as yet no clear cut method that allows distinction of capacitated from noncapacitated spermatozoa (6). Capacitation does not occur synchronously in spermatozoa (7). In addition, capacitation is transient and already capacitated spermatozoa cannot be capacitated again (8). These complexities in in vitro capacitation makes it difficult to appropriately interpret in vitro studies. This review mainly focuses on three aspects of molecular changes that occur during capacitation: concentration of intracellular calcium and other ions, changes in lipid distribution and composition and changes in protein phosphorylation and kinases activities (Fig. 1).
Figure 1. Schematic representation of the main events occurring under conditions leading to capacitation in vitro. Changes in membrane permeability to several ions have been described, among these Ca2+ and bicarbonate (HCO3-), whose influx increase during capacitation, have been reported to exert a primary role in the process. Membrane fluidity increases due to the loss of cholesterol from the membrane which may be accelerated by the extracellular presence of serum albumin (BSA). A time-dependent increase of tyrosine phosphorylation of proteins has also been described. In mouse sperm, the increase of tyrosine phosphorylation is primarily dependent on the increase in Ca2+ and bicarbonate, which, in turn, activate adenyl cyclase (AC) with increased generation of cAMP and subsequent activation of protein kinase A (PKA). PKA activation leads to the activation of sperm tyrosine kinase(s). On the contrary, in human sperm Ca2+ inhibits, rather than increases, tyrosine phosphorylation during capacitation, indicating the existence of different regulatory pathways of this process among different species. Other possible physiological modulators of tyrosine phosphorylation in human sperm during capacitation are Talpha1 (present in seminal plasma and in oviductal fluids) and reactive oxygen species (ROS) that may generate from spermatozoa. Remodelling of sperm membrane phospholipids and activation of phospholipases (PLA2 and PLCgamma1) have also been shown: in particular, increased synthesis of phosphatidylcholine (PC) from phosphatidyl-ethanolamine, phosphatidylinositol (PIP) and lyso-phosphatidylcholine (lyso-PC) have been documented. In the mouse a translocation of PLCgamma1 from a soluble to a particulate fraction during capacitation has been demonstrated. A possible activation of protein kinase C (PKC) has also been reported. =>, stimulatory pathway; =>, inhibitory pathway.
2.1. Modification in concentration of intracellular calcium and other ions during capacitation
Modification of intracellular concentration of calcium ions (Ca2+) is the most fully characterized biochemical event during capacitation. An increase in the concentration of Ca2+ during capacitation has been demonstrated in several mammalian species (9-14) including human (3,15). Extracellular Ca2+ appears to be necessary for the completion of capacitation of spermatozoa in vitro (15-20) and the increase of intracellular Ca2+ is required for capacitation (19-20). In spermatozoa the intracellular Ca2+ is regulated by the Ca2+-ATPase (acting as a Ca2+ extrusion pump) (21), Ca2+/H+ exchanger system plus Na+/Ca2+ antiporter (acting as Ca2+ entrance systems) in the plasma membrane (22), and possibly, by the intracellular Ca2+ stores (23-24). It has been hypothesized that modulation of the activity of the Ca2+-extrusion system, in particular Ca2+-ATPase, occurs during capacitation and leads to an increase in intracellular Ca2+(21-25). Drugs such as quercetin, that inhibit Ca2+-ATPase accelerate capacitation (21-22,25-27) whereas calmodulin inhibitors such as trifluoperazine and naphtalensulfonamide (W-7) enhance capacitation (21-22,25-27). A Na+/Ca2+ exchanger is present in mammalian sperm, however, its role in controlling intracellular Ca2+ during capacitation is not clear. A low molecular weight protein, caltrin, associated with ejaculated bull spermatozoa, which inhibits the Na+/Ca2+ exchanger maintains the intracellular Ca2+ at low levels (27). In the female genital tract, conformational changes appear to allow this protein to stimulate the exchanger and induce a Ca2+ in/Na+ out movement (27). Ca2+ channels have been demonstrated in mammalian sperm (2, 28-29), however, their role in modulating sperm intracellular Ca2+ during capacitation is controversial (22). The increase in intracellular Ca2+ during capacitation is a slow process reaching a plateau after 90-120 minutes from the beginning of the process (3). This is inconsistent with the mode of action of Ca2+ channels that usually mediate a rapid influx of Ca2+ suggesting that these channels are involved in the influx of Ca2+ preceding the acrosome reaction (22).
Besides Ca2+, intracellular K+ (30), Na+ (31) and Cl- (22) concentrations have been shown to be modulated during capacitation. Moreover, Na+,K+-ATPase activity increases in the hamster spermatozoa during this process (27). Activation of this pump is expected to be associated with a decrease in the intracellular concentration of Na+. However, paradoxically, increase in the intracellular concentration of this ion has been reported (22, 31). Such increase of intracellular Na+ appears to be important for capacitation, since the Na+ ionophore, monesin, promotes this process in mouse sperm (22).
A rise in intracellular pH has been reported during capacitation of bovine (32) but not hamster (10) sperm. However, the role of pH is not apparent since the increase in pH in spermatozoa does not accelerate capacitation (22). On the other hand, an important role for bicarbonate in the capacitation has been shown in several mammalian species (33-36,19). The action of bicarbonate may be related to its role in stimulation of adenylate cyclase activity rather than pH buffering capacity (37-38). The intracellular concentration in zinc ion decreases in the acrosome of hamster spermatozoa during capacitation (39). In addition, incubation of spermatozoa in a zinc-containing medium inhibits capacitation (39). These findings suggest that zinc may play a role in destabilization of plasma membrane during capacitation (39).
2.2. Changes in membrane lipids and phospholipids during capacitation
Changes in the distribution and composition of plasma membrane lipids and phospholipids are another important feature of sperm capacitation. These changes lead to an increase in the membrane fluidity (1). Among these changes is cholesterol removal which leads to a decrease in the cholesterol:phospholipid molar ratio in the sperm plasma membrane (40-46). It has been suggested that serum albumin may be responsible for the cholesterol removal (46-48). However, such an effect may simply be induced by washing of the spermatozoa (49). Purified lipid transfer protein, a serum protein present in the follicular fluid which is able to increase sperm capacitation (50), may provide the physiological mechanism for in vivo capacitation.
The amount of phospholipids does not appear to change considerably during capacitation (1). However, capacitation is associated with an increase of phospholipid methylation and increased synthesis of phosphatidylcholine from phosphatidylethanolamine (51). Incubation of spermatozoa in capacitating conditions, in the presence of bicarbonate, does not alter phospholipid distribution (52). Such condition, however, strongly inhibits phospholipid transfer, but leads to a slow increase of phosphatidylcholine concentration in the inner leaflet of the membrane (52). Levels of phosphatidylinositol and lyso-phosphatidylcholine increase during capacitation in vivo in porcine sperm (53). In view of the fusogenic property of lysophospholipids, increase in their relative amount may prepare the sperm for the acrosome reaction. Phospholipase A2 is an enzyme that generates lysophospholipids through hydrolysis of phospholipids (54). This enzyme seems to be implicated in sperm capacitation (55). Moreover, the presence of another enzyme, phospholipase Cgamma1 (PLCgamma1), that leads to hydrolysis of phosphoinositides has been demonstrated in mouse spermatozoa (56). During capacitation mouse spermatozoa PLCgamma1 translocates from a soluble to a particulate fraction (56). This translocation may be required for establishment of contact between this enzyme and activated membrane protein tyrosine kinases in the mammalian spermatozoa (see later). In addition, capacitation is associated with a shift in the electrophoretic mobility of PLCgamma1 (56). Such slower electrophoretic mobility may be due to tyrosine phosphorylation and activation of PLCgamma1 (57-58).
2.3. Changes in protein phosphorylation and protein kinase activity during capacitation
Generally, protein phosphorylation is stimulated by activation of kinases (59). The best studied kinases involved in capacitation are Ca2+-calmodulin activated kinases, cAMP-dependent kinases (PKA), calcium and phospholipid activated protein kinase (PKC), which induce phosphorylation of proteins in serine and threonine residues, and tyrosine kinases, which phosphorylate proteins in tyrosine residues.
2.3.1. Involvement of PKA in capacitation
Adenylcyclase activity and synthesis of cAMP increase during capacitation of mouse sperm (60-62). cAMP agonists and phosphodiesterase inhibitors counteract the inhibitory effect of glucose on capacitation of bovine spermatozoa (63). Pentoxifylline, which promotes an increase in cAMP, induces capacitation (64). These findings suggest that PKA may be involved in the process of capacitation. A more direct evidence for the involvement of cAMP-dependent kinases in the process of capacitation has been recently reported (38). Two different inhibitors of PKA inhibited capacitation (38). Moreover, the process of capacitation could be induced by addition of biologically active cAMP agonists (38). The increase in concentration of cAMP in spermatozoa is dependent on presence of Ca2+ and bicarbonate in the extracellular medium (65). Inhibitors of PKC and Ca2+/calmodulin dependent protein kinases, however, do not seem to have any effect on the concentration of cAMP (38).
2.3.2. Involvement of PKC in capacitation
Presence of PKC in mammalian spermatozoa and its role in sperm motility and the process of acrosome reaction are documented (66-68). However, the role of this enzyme during capacitation is poorly understood. Stimulation of PKC with phorbol esters accelerates the process of capacitation (69). This effect was inhibited by PKC inhibitors, suggesting that PKC may be involved in capacitation (69). In addition, PKC may also be involved in epidermal growth factor- induced capacitation (70). However, these studies were performed using phorbol esters as PKC inducers, and non specific effects of these tumor promoters on other kinases cannot be excluded. PMA and diacylglycerol stimulate the acrosome reaction only in spermatozoa that have been previously capacitated (68,71). Such evidence favours the concept that PKC pathway is implicated in capacitation.
2.3.3. Involvement of tyrosine phosphorylation in capacitation
The first evidence for the presence of tyrosine phosphorylated proteins in mammalian spermatozoa dates back to 1989 (72). Using anti-phosphotyrosine antibodies, Leyton and Saling identified three different phosphoproteins at 52, 75, and 95 kDa in the mouse spermatozoa (72). The 95 kDa protein was tyrosine phosphorylated under all experimental conditions and including interaction of spermatozoa with solubilized ZP proteins (72). The 75 kDa and 52 kDa proteins were phosphorylated only in capacitated spermatozoa and may represent capacitation-specific markers (72). Later on, similar tyrosine phosphorylation pattern was reported in human spermatozoa (73-75). Phosphorylation on tyrosine residues occurs during capacitation, interaction of spermatozoa with zona proteins and with progesterone (73-76). The increase in tyrosine phosphorylation during capacitation is time-dependent (Fig. 2).
Figure 2. Western blot analysis of the reactivity of the anti-phosphotyrosine antibody with human sperm proteins at various times of in vitro capacitation. NC indicates capacitation at 0 min. Tyrosine phosphorylated proteins were revealed using an anti-phosphotyrosine polyclonal antibody followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit antibody and stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate. Molecular weight markers (kDa) are indicated to the left of the blot. The arrows indicate the 97 and the 75 kDa phosphoproteins. From Luconi et al. (74) with permission.
Such phosphorylation has functional consequence. Incubation of spermatozoa with antiphosphotyrosine antibodies or inhibition of tyrosine kinase activity inhibited zona-free hamster egg penetration (73), prevented acrosome reaction (77) and blocked fertilization (77). However, erbstatin, a potent inhibitor of tyrosine kinase, did not inhibit capacitation measured as the ability of spermatozoa to respond to progesterone (20). Immunofluorescence labelling of phosphotyrosine residues, indicated that capacitation as well as exposure to zona proteins increased the degree of tyrosine phosphorylation in each spermatozoon and particularly increased the number of sperm cells phosphorylated in the acrosomal region of the sperm head (73). The site of phosphotyrosine-specific fluorescence shifted from the tail of non-capacitated sperm to the acrosome of capacitated spermatozoa (73). There are only a few membrane proteins on the sperm cell that are phosphorylated on tyrosine residues. Among these, the highest degree of tyrosine phosphorylation was found in a protein of 95-97 kDa (73-75). A phosphoprotein in this molecular weight range was found to be the sperm receptor for ZP3, ZRK. This protein has been characterized, partially cloned and sequenced. Sequence shows a 55% homology with the receptor-like protein tyrosine kinase c-eyk (75) and 97-100% homology with the proto-oncogene c-mer (78). This protein has been found only in mature spermatozoa and testicular germ cells (75). A mouse sperm tyrosine phosphorylated protein, migrating at 95 kDa in non reducing conditions, has been sequenced (79). This protein shows complete amino acid homology to a mouse hepatoma hexokinase (HK1) (79). Indeed, the purified sperm protein reacted with an antiserum to the purified rat brain hexokinase type 1 (79). This protein could also be immunoprecipitated with an anti-phosphotyrosine antibody (79). The tyrosine phosphorylated form of HK1 is apparently present only in sperm and testis (80). HK1 is located on the sperm head, has an extracellular domain and behaves like an integral membrane protein (80). Although this protein appears to be phosphorylated, its level of phosphorylation does not change during capacitation in the mouse (19,38). On the other hand, an antibody against ZRK, immunoprecipitated a 95 kDa phosphotyrosine containing protein in human spermatozoa that could not be recognized by an antibody to hexokinase (75). Similarly, a different anti-ZRK antibody, LL95, that bound to the acrosomal region of mouse sperm and which could mimic the effects of ZP3 did not recognize the hexokinase (81). In addition, the anti-hexokinase antibody, although bound to sperm tail region, failed to demonstrate any effect on sperm-zona binding or to stimulate the acrosome reaction (81). Taken together, these data suggest that p95 hexokinase is not involved in sperm-oocyte interaction (81).
During capacitation, the role of several regulating agents in tyrosine kinase activity and protein tyrosine phosphorylation has been studied. In spermatozoa of mouse epidydimus, the increase of protein tyrosine phosphorylation during capacitation appeared to be dependent on the presence of BSA, calcium and bicarbonate in the medium (19). The capacitation-inducing activity of these factors was completely dependent on the generation of cAMP (38,82). Addition of cAMP agonists could restore capacitation-induced protein tyrosine phosphorylation in the absence of any of these factors (38). In human spermatozoa, the requirement of calcium ions in the medium for capacitation is different from that observed in the mouse. Depletion of Ca2+ in the medium potentiates, rather than inhibits, the protein tyrosine phosphorylation during capacitation of human spermatozoa (20). Presence of calcium chelating agents in the capacitating medium is incapable to decrease such enhanced tyrosine phosphorylation. Moreover, increasing intracellular calcium with calcium ionophores leads to a decrease in the tyrosine phosphorylation of sperm proteins (20). Two thymosin peptides, Talpha1 and Tß4 were detected in the seminal plasma of men and in the follicular fluids of women (83): the rate of penetration of zona-free hamster ova by human spermatozoa was increased by Talpha1 but not Tß4 (84). A significantly higher concentration of Talpha1 was present in the seminal plasma of fertile rather that infertile men. On the contrary, Tß4 was higher in seminal plasma of infertile as compared to fertile men (84). Talpha1 increases the capacitation of human spermatozoa by enhancing tyrosine phosphorylation of several sperm proteins (84). Two of these proteins with molecular weights of 95 and 51 kDa bind to the zona pellucida (84). The stimulatory effect of Talpha1 on phosphorylation is exerted only in membrane protein extracts of non-capacitated spermatozoa (85). Talpha1 has no effect on protein tyrosine phosphorylation of capacitated spermatozoa (85). Besides the 95-97 kDa protein indicated above, a 51 kDa protein designated fertilization antigen-1 (FA-1), is considered to be a receptor tyrosine kinase and plays an important role in capacitation (86-88). Treatment of human spermatozoa with an anti-FA-1 monoclonal antibody reduced protein phosphorylation including tyrosine phosphorylation of both 95 and 51 kDa proteins (85).
Tyrosine phosphorylation of sperm proteins is greatly enhanced under oxidizing conditions, and reduced under reducing conditions (89). Moreover, biological response to progesterone is significantly enhanced by stimulation of reactive oxygen species (ROS) generation during capacitation and inhibited by scavengers of hydrogen peroxide (90).
Presence of other kinases, including c-ras (91), EGF-receptor tyrosine kinase (92), and the cell-cycle specific cyclin, cdc-2 (93), have been demonstrated in mammalian spermatozoa. The role of these enzymes in capacitation is not yet clear. However, the cdc-2 serine/threonine protein kinase, is expressed at a higher level in capacitated spermatozoa, suggesting a possible role of this protein in the process (93).
3. THE ACROSOME REACTION (AR): BACKGROUND
The sperm acrosome, a Golgi-derived structure forming a cap over the anterior region of the nucleus, contains many hydrolytic enzymes (1). Acrosome consists of an anterior cap and a posterior region called equatorial segment (94). During capacitation, the spermatozoa acquires the ability to penetrate the cumulus oophorus and to bind to the zona pellucida. This binding triggers AR which consists of development of multiple fenestrations between the outer acrosomal membrane and the plasma membrane of the spermatozoa (1). This interaction leads to the release of the enzymatic content of acrosome and to the exposure of the enzymes bound to the inner membrane adjacent to the nuclear envelope (1) (Fig. 3).
Figure 3. Diagram illustrating the process of acrosome reaction. (A) Intact spermatozoon. (B) Acrosome reaction in progress: fusion and fenestration of plasma and acrosomal membrane allows the release of acrosomal contents (hydrolyzing enzymes). (C) Acrosome-reacted spermatozoon. From (101) with permission.
This exocytotic process, involves the anterior region of sperm head and is not extended beyond the equatorial segment (1). In the absence of any specific stimuli, human spermatozoa can undergo acrosome reaction at a low level (95). It has been suggested that self aggregation of sperm receptor for zona pellucida may account for this spontaneous acrosome reaction (96). According to another hypothesis, the Na+ and/or Ca2+- pumping mechanisms become less efficient with time (due to depletion of ATP, for example) (1). This would result in a gradual increase in intracellular Ca2+ and pH which leads to spontaneous acrosome reaction (1). Molecules such as ATP (97-98) and phosphodiesterase inhibitors like caffeine and pentoxifylline (99-100) and progesterone (101) stimulate the AR in vitro. ZP3, a sulphated glycoprotein in the zona pellucida of mammals, is the egg protein that physiologically induces AR (1). Other potential stimuli of AR in vivo include the oviductal and follicular fluids as well as several proteins in the cumulus oophorus matrix (Table 1). It is conceivable that the high concentration of progesterone and other steroids in the cumulus oophorus induces AR during the fertilization process. Solubilized zona pellucida and progesterone synergistically induce AR in the mouse and porcine spermatozoa in vitro (110,111). Progesterone in the cumulus matrix may "prime" spermatozoa subsequently to their interaction with ZP3 and before they have fully undergone the AR (110). Higher binding of human spermatozoa pre-treated with human follicular fluid to the zona pellucida is consistent with this hypothesis (112).
| AGONIST | REF |
|---|---|
| Zona pellucida protein (ZP3) | 102 |
| Serum albumin | 103 |
| Epidermal growth factor | 104 |
| Atrial natriuretic peptide | 105, 106 |
| Platelet-activating factor | 107, 108 |
| Progesterone & 17alphaOH-progesterone | 101, 109 |
One of the first events that occurs in spermatozoa following stimulation with ZP3 and progesterone is receptor aggregation (102, 113-114). This is followed by a cascade of downstream membrane and cytosolic signaling factors involved in induction of AR (Fig. 4). Among them, the roles of calcium, phospholipases and protein kinases are discussed below.
3.1. Increase in intracellular calcium during acrosome reaction.
Calcium plays a central role in receptor-mediated response and membrane fusion processes in spermatozoa (1,115). Calcium ionophores are indeed the most widely used non physiological inducers of AR (1). However, AR can be induced in the absence of extracellular calcium with some agonists (97,108,116), whereas extracellular calcium in the millimolar range is essential to the exocytosis induced by ZP3 (25).
Presence of calcium channels was originally shown in the plasma membrane of sea urchin spermatozoa (117). Using the fluorescent probe fura-2, the amount of intracellular calcium in response to addition of ZP3 was studied in mammalian spermatozoa (28, 118). Addition of ZP3 led to a rapid (2-5 min) increase in the amount of intracellular calcium. This was followed by a plateau phase lasting 10-15 min (28, 118). Acrosome reaction occurred during the sustained phase of calcium increase (119). Activation of calcium channels by ZP3 involves pertussin toxin sensitive GTP-binding proteins (118). Ca2+ influx stimulated by ZP3 involves specific L-type voltage-dependent plasma membrane Ca2+-channels (28). Ca2+-channel blockers blunt the increase in the intracellular calcium and the AR induced by zona pellucida (28).
Increase of intracellular Ca2+ is associated with an efflux of H+ and a rise in intracellular pH (118). ZP3 also activates non-selective cation channels that lead to membrane depolarization and opening of voltage-sensitive Ca2+-channels (119).
Similar to ZP3, progesterone and follicular fluid induce an influx of calcium in human sperm (120-122). However, in this case, pertussin toxin sensitive GTP-binding proteins and voltage-sensitive calcium channels do not appear to be involved (101,123). Extracellular calcium seems to be required for induction of exocytotic event by progesterone (101). Some data suggest that calcium may be stored in mammalian sperm (23-24,124). For example, thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+-ATPase induced an influx of calcium (23) and led to AR in the capacitated human sperm (124-125). These effects were dependent on the presence of extracellular calcium (23,125). According to one hypothesis, thapsigargin induces release of calcium from intracellular stores (126). This, in turn, leads to a massive influx of extracellular calcium (126). Spermatozoa do not posses endoplasmic reticulum. Therefore, it has been postulated that such storage sites may exist in the nuclear envelope or in the outer acrosome membrane as suggested by recent data (23, 124-125). For example, the newly identified and characterized IP3 receptors on the outer acrosome membrane of rat sperm may play a role in Ca2+ storage (124). Similarly, calreticulin, a calcium binding endoplasmic reticulum protein involved in calcium release, has been recently described in the rat acrosome (127).
Calcium may play a role in the fusion events in the sperm membrane (128). Using a pyroantimonate-osmium fixation technique, the temporal and spatial location of intracellular calcium granules was monitored during acrosome reaction in ram spermatozoa (128). Ca2+ is initially associated with the outer acrosomal membrane. As the process progresses, Ca2+ associates with the fusion sites between the outer acrosomal membrane and the plasma membrane anteriorly to the equatorial segment. At later stages, Ca2+ is localized in both post acrosomal dense lamina and on outer acrosomal membrane under the equatorial segment. These findings suggest that Ca2+ may be implicated in the fusion process (128)
Fluxes of Na+ (25,129), Cl- (130-134), bicarbonate (135) and H+ (25,119,129) occur during AR, suggesting that, besides Ca2+ , other ions may be implicated in the process of fusion of the outer acrosomal membrane and the plasma membrane of spermatozoa.
3.2. Phospholipases activation during acrosome reaction.
It was shown earlier that during AR in boar spermatozoa, the amount of diacylglycerol and free fatty acids increases (136). This finding was consistent with activation of phospholipases. In fact, increase of intracellular Ca2+ stimulated by ionophores and progesterone leads to activation of phospholipase C in human spermatozoa (120,137-138). Such activation leads to an intracellular increase in the amount of inositol trisphosphate and diacylglycerol. Similarly, ZP3 activates the phosphoinositide specific enzyme phospholipase C (55) by virtue of tyrosine phosphorylation and leads to its translocation from cytosol to particulate fractions (55). Presence and activation of phosphatidylcholine-specific phospholipase C during AR has been also demonstrated in the mammalian spermatozoa (139-140).
Similarly, the AR induced by ionophores and progesterone leads to the activation of phospholipase A2 (141-143). This activation is associated with generation of lipid metabolites, such as arachidonic acid and lysophospholipids. Phosphatidylcholine, lysophospholipids, and unsaturated fatty acids, such as arachidonic acid, are potent inducers of AR (144-149) and may be implicated in the fusion process that occurs during AR. Moreover, lysophosphatidylcholine generated from PLA2 activation, may act as a substrate for generation of platelet-activating factor, in the mammalian spermatozoa (150-151). This phospholipid, that is synthesized during AR (143), may further enhance AR and sperm motility (108,152-155).
During AR, phospholipase D is activated and phosphatidic acid is generated (156-158). However, activation of phospholipase D represents a late event in the AR process, and does not appear to substantially contribute to this event (157-158).
3.3. Activation of protein kinases during acrosome reaction.
During AR, activation of protein kinases (serine-threonine kinases as PKC, PKA and PKG, and tyrosine kinases) is downstream to the production and/or activation of early second messengers. Preliminary evidence for the involvement of PKA in the AR, demonstrated that adenylate cyclase activity and cAMP generation increases during this process (159). Adenylcyclase agonist, forskolin, and the cAMP analogue, dibutyryl cAMP, induce AR in a dose-dependent manner (159-161). These findings suggest that PKA may be involved in AR. PKC activators such as phorbol esters and synthetic DAG, induce acrosome reaction (68,71). Controversial data were reported regarding the strict dependence of this effect on presence of extracellular calcium (68-71). Bielfeld et al. (162), showed that stable agonists of cAMP and cGMP, as well as phorbol esters can induce AR in capacitated spermatozoa. This induction did not require presence of extracellular calcium. Similarly, stimulation of kinase activity by zona pellucida does not require extracellular calcium (162). Induction of AR by solubilized zona pellucida was partially reduced by pretreatment with inhibitors of PKA, PKC and PKG tested separately, while combination of them caused a significantly greater inhibition. These results suggest an important, concomitant role for PKA, PKC and PKG in human ZP-induced acrosome reaction (162). Combination of PKA and PKC stimulators at their ED50 induced an AR equivalent to that obtained with a single stimulator at its EDmax, while the combination of the two agents at their EDmax was not additive (161). Taken together these results suggest that although the two signalling pathways are partially independent, cross-talks between these pathways are likely to occur. Inhibitors of PKC counteract the effect of progesterone on AR (163).
ZP3 induced an increase in tyrosine phosphorylation of sperm proteins (72,75,77). A major tyrosine phosphorylated protein in mammalian spermatozoa was 95-97 kDa (72-76). This protein appears to undergo autophosphorylation during capacitation and in response to inducers of AR such as ZP3 (72,75) and progesterone (74,76,164). In addition to the 95 kDa receptor protein for ZP3 (75) other sperm binding proteins have been characterized. These include galactosyltransferase (165) and sp56 (166-167) in the mouse, and the FA-1 antigen in the human (168). The inhibitors of tyrosine kinase, genistein and Tyrphostin 47, block ZP3-induced AR (77). Moreover, the tyrosine kinase inhibitor, tyrphostin A48, and pertussis toxin, both inhibit the ZP3-induced calcium influx in the mouse spermatozoa (169). Using a similar pharmacological approach, Tesarik et al. (76) and Luconi et al. (74) showed involvement of tyrosine kinases in the progesterone-mediated acrosome reaction. Activation of tyrosine kinase seems to be involved in the plateau phase of increase in the amount of intracellular Ca2+ in response to progesterone (170-171) (Fig. 4).
Figure 4. Diagram illustrating the main signaling pathways so far described to be involved in the process of acrosome reaction in response to zona protein 3 (ZP3). Following interaction with the agonist, aggregation of receptors for ZP3 (ZRK) induces TK activation (which increases protein tyrosine phosphorylation) and autophosphorylation of the receptor. A guanine nucleotide binding protein (G protein) transduces the signal interacting with membrane-bound enzymes like phospholipase C (PLC) and adenylate cyclase (AC). Activation of these two enzymes leads to increased generation of the second messengers cyclic adenosine monophosphate (cAMP), inositol trisphosphate (IP3) and diacylglycerol (DAG). A consequence of the increase of second messengers is the activation of protein kinases such as cAMP-dependent kinase (PKA) and Ca2+ and phospholipid-dependent kinase (PKC) with increased protein phosphorylation. cAMP-dependent influx of sodium (Na+) has been reported. IP3 may increase intracellular Ca2+ by liberation of the ion from intracellular Ca2+ stores (not demonstrated in sperm). The increase of intracellular Ca2+ consequent to activation of ZP3 receptors is completely due to influx from the extracellular medium, is dependent on activation of G proteins, involves voltage-dependent Ca2+ channels and is accompanied by an efflux of H+ which determines a rise of intracellular pH (pHi). Partial dependence of Ca2+-influx from TK activation has been reported. Ca2+-dependent activation of phospholipase A 2 (PLA2) and phospholipase D (PLD) [with increased generation of other second messengers as arachidonic acid (AA), lyso-phosphatidylcholine (LC) and phosphatidic acid (PA) from membrane phospholipids (Phlp)] have also been described to occur during acrosome reaction.
Reactive oxygen species may be involved in the regulation of tyrosine phosphorylation in human spermatozoa (89-90). It has been suggested that the increase in tyrosine kinase activity by free radical during capacitation in human spermatozoa improves the response of human spermatozoa to progesterone stimulation (90).
In vitro capacitation has allowed determining a number of biochemical events that occur in human spermatozoa. It is not clear whether the same or similar events occur during capacitation that takes place in vivo. However, progesterone and platelet-activating factor, present in the follicular fluid and/or cumulus matrix, could facilitate or prime the acrosome reaction in vivo. The responsiveness of spermatozoa to progesterone might be used as a routine clinical test (172-175). Based on such a test, males can be separated into those with normal or reduced sperm response to progesterone (175). Based on such data, the decision can be made to either perform in vitro fertilization or to resort to a more invasive technique such as intracytoplasmatic sperm injection (ICSI).
We thank Prof. Mario Serio (Endocrinology Unit, University of Florence) for revision of the manuscript.
Supported by Consiglio Nazionale delle Ricerche (CNR, Rome - Progetto FATMA), Ministero dell'Universita' e della Ricerca Scientifica and Regione Toscana.
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