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 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).