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

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).

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 (pH_i). 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).