Human Reproduction vol 6 no.9 pp. 1265-1274, 1991
OPINION
Human sperm capacitation and the acrosome reaction*
L.J.D.Zaneveld1, C.J.De Jonge, R.A.Anderson and S.R.Mack
Key words: spermatozoa/acrosome/human/G-proteins/protein phosphorylation
Departments of Obstetrics and Gynecology, Biochemistry and Physiology, Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA
Introduction
'To whom correspondence should be addressed
A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phosphoupid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion. •Presented at the 2nd Dusseldorf Symposium on Interactions in Reproductive Medicine, November 18-20, 1990. Prepared for publication by N.J.Alexander, G.Freundl and V.Insler.
© Oxford University Press
The requirement for epididymal or ejaculated spermatozoa to undergo modifications before they are able to fertilize the oocyte was first described in 1951. Numerous articles have addressed the biochemical, physiological and morphological aspects of capacitation and the acrosome reaction, as summarized in a variety of reviews (for example, McRorie and Williams, 1974; Hoskins and Casillas, 1975; Bedford and Cooper, 1978; Stambaugh, 1978; Meizel, 1978, 1984, 1985; Green, 1978; O'Rand, 1979; Bhattacharyya and Zaneveld, 1982; Rogers and Bentwood, 1982; Clegg, 1983; Monroy and Rosati, 1983; Chang, 1984; Hinrichsen-Kohane et ai, 1984; Austin, 1985; Langlais and Roberts, 1985; Tesarik, 1986; Peterson etal., 1987; Wasserman, 1987; Yanagimachi, 1988; Kopf, 1988, Garbers, 1989; Garbers and Kopf, 1989; Shur, 1989; Saling, 1991; Zaneveld and De Jonge, 1991). In spite of the extensive literature, there is almost as much confusion on the subject now as there was two decades ago. This confusion is caused by the many factors and conditions that can influence capacitation and/or the acrosome reaction under artificial conditions and the uncertainty as to which are physiological. The observations often appear to be unrelated to each other so that it has been difficult to combine the data into a unifying hypothesis. In recent years, however, much knowledge has accrued regarding the functional activity of membranes in somatic cells. It is likely that this knowledge also can be applied to spermatozoa. The following discussion is based on four hypotheses: (i) One or more of the lytic enzymes of the sperm acrosome has an essential role in the penetration of the spermatozoon through one or more of the layers surrounding the oocyte. (ii) Through the processes of capacitation and the acrosome reaction, the activation/release of the lytic enzyme(s) is controlled so that it does not occur until the spermatozoon reaches the target layer(s) of the oocyte. (iii) At least part of capacitation involves the biochemical modification of the sperm head membrane(s), allowing the acrosome reaction to occur with the appropriate oocyte signal(s). (iv) The acrosome reaction is a type of exocytosis. Due to limitations of space, only some of the more recent references that cannot be found in the reviews (see above), are cited. This review focuses on the human. 1265
L.J.D.Zaneveld et al.
Physiological importance of capacitation and the acrosome reaction The function of the spermatozoon is to deliver DNA into the oocyte. In order for this to happen, spermatozoa first pass through the female genital tract and then through the various oocyte investments, namely the cumulus oophorus, the corona radiata, the zona pellucida and the vitelline membrane (oolemma). The cumulus oophorus and the corona radiata are layers of follicle (granulosa) cells. The cumulus cell matrix consists mostly of hyaluronic acid. The zona pellucida consists of glycoproteins, the vitelline membrane is the plasma membrane that surrounds the oocyte. The fertilizing spermatozoon enters the oocyte while these layers are intact. To do so, the spermatozoon uses its forward progressive motion and lytic enzymes. The latter digest a hole through their target layers in front of the spermatozoon. As their function necessitates, the enzymes are associated with the most anterior portion of the sperm head: the sperm acrosome. The acrosome is a flattened, membrane-bound vesicle that surrounds the anterior portion of the sperm nucleus. Because of its flattened appearance, the acrosome can be divided into three parts: (i) the inner acrosomal membrane (IAM) that overlies the nuclear membrane; (ii) the outer acrosomal membrane (OAM) which represents the outer layer of the vesicle; and (iii) the acrosome proper, located between the IAM and the OAM. The IAM and OAM join at the equatorial segment. The entire spermatozoon, including the acrosome, is surrounded by the plasma membrane. A small area of cytosol is present between the OAM and plasma membrane (cytosolic space). The area inside the acrosome is referred to as the intra-acrosomal space. The acrosome may have similarities to a secretory vesicle that contains digestive enzymes which are released through the process of exocytosis when the cell receives the appropriate signal ('regulated pathway of secretion'). The most intensively investigated acrosomal enzyme is acrosin, a serine proteinase. Numerous studies have indicated that acrosin is essential for fertilization. The enzyme appears to have a role in sperm binding to the zona pellucida, sperm penetration (lysis) of the zona pellucida and the acrosome reaction (see below). Acrosin is almost entirely present in an inactive form (proacrosin). Proacrosin is primarily associated within the acrosome proper and IAM although some proacrosin can be found on the plasma membrane. It can be speculated that surface proacrosin is involved in the binding of the spermatozoon to the zona pellucida, whereas the intra-acrosomal proacrosin is involved in the acrosome reaction and zona lysis. Intra-acrosomal proacrosin is converted to acrosin and exposed by the partial or complete removal of the plasma membrane and OAM, before acrosin can exert its activity. This is probably the reason why morphologically unmodified (non-acrosome reacted) spermatozoa cannot penetrate the zona pellucida. The following model utilizes acrosin as an example but the same model probably applies to other sperm-head enzymes. For the correct utilization of the enzyme and to ensure that this highly lytic enzyme does not damage non-target tissue, activation of proacrosin and release of acrosin must be coordinated with sperm approximation to the oocyte. We propose that such selective exposure is controlled by sperm capacitation 1266
and the acrosome reaction. Capacitation can be defined as the biochemical modifications that are required for the acrosome reaction to occur in response to the appropriate stimulus. The acrosome reaction involves the localized fusion of certain sections of the plasma membrane with the OAM, the lysis/disappearance of these fused areas, the formation of vesicles by the remaining PM and OAM, the dispersal of these vesicles and the release of the acrosomal contents. After the acrosome reaction, only the IAM remains attached to the spermatozoon. No proacrosin activation occurs during capacitation but as the acrosome reaction takes place, a large proportion of the proacrosin is converted to acrosin which is then released. Physiologically, it would be most advantageous if the acrosome reaction is completed, i.e. if acrosin is released, after the spermatozoon binds to the zona pellucida so that the enzyme exerts its activity specifically at the site where sperm binding takes place. Presently, it is argued whether the acrosome reaction occurs before or after sperm binding to the zona. Acrosomereacted spermatozoa have not only been detected on the zona pellucida but also in the follicle cell layers of the oocyte. Furthermore, the acrosome reaction can be stimulated by both zona glycoproteins and follicle (cumulus) cell secretions. Therefore, it is reasonable to assume that the acrosome reaction can be initiated while the spermatozoon moves through the follicle cell layers. Since such transport is fairly rapid, the acrosome reaction is most probably not completed until after the spermatozoon binds to the zona pellucida. However, in the case that sperm transport through the follicle cell layer is delayed, the acrosome reaction may be completed before the spermatozoon reaches the zona pellucida. Whether such spermatozoa can penetrate the zona pellucida remains to be established but they are probably disadvantaged (e.g. Liu and Baker, 1990). Some proacrosin remains bound to the IAM after the acrosome reaction is completed. Preliminary evidence tends to suggest that the IAM-bound proacrosin is activated as the spermatozoon passes through the zona, thus favouring the formation of the penetration slit in front of the spermatozoon. It also appears that capacitation and the acrosome reaction have to occur before spermatozoa can fuse with the vitelline membrane (the basis of the zona-free hamster oocyte penetration assay). Acrosin and /or other proteolytic enzymes may be involved in vitelline membrane penetration since proteinase inhibitors prevent this process.
Capacitation Location and control It appears that spermatozoa can become capacitated in any of the tubular organs of the female genital tract (except possibly the vagina) and in the cumulus oophorus. However, the efficacy of the sites may vary. Sperm transport through the female genital tract can occur quite rapidly (times as short as 15 —30 min have been reported in the human), whereas capacitation requires a much longer time ( 3 - 4 h is estimated for the human). Thus, it can be speculated that the fertilizing spermatozoon gradually undergoes capacitation as it moves through the female genital tract but does not complete the process until after it has entered the cumulus oophorus. This is physiologically beneficial because
Human sperm capacitation and the acrosome reaction
the spermatozoon remains unresponsive to signals inducing the acrosome reaction until it approaches the zona pellucida, preventing a premature acrosome reaction. However, under conditions of long-term storage, the capacitation process can be initiated and, presumably, even be completed in any one of the tubular genital organs. Spermatozoa only become capacitated in the oestrogen- and not the progesterone-stimulated genital tract. The effect of these steroids is not directly on the spermatozoon and is probably mediated via the genital tract. The capacitation factors in the genital tract secretions remain to be identified although several that act in vitro (see below) are also known to be present in genital tract secretions. Identification of the physiologically active factors is problematic since the incubation of spermatozoa in isolated uterine or Fallopian tube secretions inconsistently induces capacitation. One explanation is that contact with the endometrium or endosalpinx is required but some evidence argues against this possibility. Mechanism General considerations The plasma membrane consists of a lipid bilayer in which numerous proteins are dispersed. As with somatic cells, the predominant lipids of the sperm bilayer are phospholipids, cholesterol and glycolipids. The cholesterol/phospholipid ratio of the human sperm plasma membrane is about unity (Mack et ai, 1986). In somatic cells, the integral membrane proteins often contain carbohydrates (i.e. are glycoproteins) and can extend from the outer (extracellular) surface of the membrane to the inner (cytosolic) surface. These proteins have important functions, including ion transport and receptor activation. The carbohydrate portions of the glycolipids and the glycoproteins are always orientated away from the the cytosolic surface of the membrane, i.e. located on the extracellular surface of the plasma membrane but on the inner surface of secretory vesicles. The carbohydrate-rich, outer surface of the plasma membrane is charged and often called the glycocalyx. It also contains absorbed glycoproteins or polysaccharides which bind via noncovalent interactions and do not extend into the lipid bilayer. Such peripheral membrane components can be removed, for instance, by high or low ionic strength solutions or extreme pH variations. In somatic cells, it is known that these peripheral components can block membrane receptors or the function of transport proteins. Removal of peripheral membrane factors Factors may exist in or on the sperm surface which block the acrosome reaction or prevent sperm binding to the zona (such binding may be required for the completion of the acrosome reaction). Acrosome reaction blockers could conceivably be peripheral membrane factors that prevent extracellular signals from interacting with sperm surface receptors, or inhibit ion channels or enzymes, and/or are intrinsic, structural factors which stabilize or otherwise alter the plasma membrane lipid bilayer so that it cannot respond. Removal of these factors is required before the acrosome reaction can occur and is an essential aspect of the capacitation process. Spermatozoa absorb testicular, epididymal and seminal fluid
components during their transport through the male genital tract and during ejaculation. When incubated in seminal plasma, capacitated spermatozoa lose their ability to undergo the acrosome reaction and to penetrate the oocyte, i.e. become 'decapacitated'. Such decapacitated spermatozoa must be 'recapacitated' before they are capable of fertilizing, thus, an early step in capacitation is probably the removal of certain inhibitory factors. The reversible binding of these factors suggests that they are peripheral membrane components and part of the glycocalyx. Many epididymal and seminal factors adhere to spermatozoa but to date only three are known to prevent fertilization reversibly. One of these is a group of relatively high molecular weight (Mr) glycoproteins (180 to 260 kDa) for which the terms 'decapacitation factor', 'antifertility factor', 'acrosomestabilizing factor' and 'acrosome-reaction inhibiting factor' have been used. It appears that these glycoproteins differ in the mechanism by which they prevent the fertilizing capacity of spermatozoa. For instance, our laboratory has shown diat one high Mr human factor prevents the acrosome reaction, whereas another prevents sperm penetration through the zona pellucida without an apparent effect on the acrosome reaction, possibily by preventing sperm binding. The mechanism of action of these gycoproteins is unknown. Their origin is primarily the seminal plasma although they can also be found in epididymal fluid. Another epididymal/seminal glycoprotein which reversibly prevents capacitation, is acrostatin. Human acrostatin has a M r of ~ 5 kDa and inhibits acrosin. Since proacrosin may be a zona binding protein and since acrosin is involved in die acrosome reaction (see further), acrostatin probably acts by inhibiting either or both of these processes. In the mouse, an epididymal acrosin inhibitor is known to prevent zona binding. The third factor is a high M r polylactosaminyl glycoside which has been isolated from mouse epididymal fluid. This factor inhibits galactosyltransferase, an enzyme on the mouse sperm membrane diat also appears to be involved in zona binding. The presence of this inhibitor in human seminal plasma and a role for galactosyltransferase in zona binding of human spermatozoa remains to be established. Any agent or method that (i) disrupts the noncovalent bonds between the peripheral membrane factors and the sperm surface; (ii) alters the carbohydrate content of the glycocalyx and/or (iii) hydrolyses the factors themselves, may cause the removal or destruction of the inhibitory agents and initiate capacitation. Thus, it is not surprising that a variety of techniques have been reported to induce capacitation in vitro, including treatment of spermatozoa with high salt concentrations, glycosaminoglycans, carbohydrases and proteinases. Albumin, a frequendy used agent for in-vitro capacitation, removes glycoconjugates from the sperm surface (e.g. Focarelli etai, 1990). Whether any of these in-vitro capacitation promoters are actually involved in the capacitation process in vivo remains to be shown. Capacitation is known to be associated with a decrease in net surface charge and a loss of carbohydrates, including sialic acid. Such changes can also be explained by the removal of peripheral glycoproteins or polysaccharides, which can contain sialic acid such as die high M r glycoprotein antifertility factors. Changes in the carbohydrate moiety of the glycocalyx may further explain the alterations in lectin binding that are observed after capacitation. 1267
LJ.D.Zanevdd et al.
Changes in membrane lipid composition If capactitation is truly a reversible process (as is generally considered) it is unlikely to be associated with major sturctural changes of the sperm membrane. In addition, structural changes may lead to a premature acrosome reaction so that such changes should optimally occur just before the acrosome reaction takes place, i.e. after the spermatozoon begins to penetrate the oocyte. Furthermore, there is no good evidence to suggest that structural modifications are required before the acrosome reaction can be stimulated. For instance, treatment of non-capacitated (washed ejaculated) human spermatozoa with calcium ionophore or dibutyryl cyclic AMP (dbc AMP), which bypass the need for surface receptor activation (see section on Acrosome reaction), causes the acrosome reaction (Anderson et al., 1990a). Although a generally held belief is that membrane destabilization and permeabilization take place during capacitation, this may not be the case. It is more likely that such modifications are part of the acrosome reaction or occur during the transition period between capacitation and the acrosome reaction. The cholesterol/phospholipid ratio is important for membrane stability. A decrease in cholesterol or an increase in phospholipids causes destabilization of the membrane and an increase in membrane permeability. Thus, it is not surprising that under invitro conditions, the acrosome reaction is stimulated by removal of cholesterol or the addition of phospholipids and that the addition of cholesterol to spermatozoa can hinder the acrosome reaction by 'hardening' the membrane. This had led to the hypothesis that the removal of cholesterol is an integral aspect of capacitation. Membrane vesicles in seminal plasma can donate cholesterol to the plasma membrane and were suggested to be 'decapacitating factors'. Cholesterol loss from membranes usually occurs via carrier proteins or enzymes. Cholesterol acceptors in blood include lipoproteins, apolipoproteins, albumin, and lecithin: cholesterol acyltransferase (LCAT). One or more of these acceptors can be found in female genital secretions and are present in media used for in-vitro capacitation and fertilization, i.e. media containing heat-inactivated blood serum, follicular fluid, albumin or egg yolk. It remains to be established whether or not the components in these media actually function as cholesterol acceptors, because they may affect the sperm membrane by another mechanism. For instance, albumin was shown to donate phospholipids rather than to remove cholesterol (Davis el al., 1980) and may cause removal of peripheral membrane glycoconjugates (see above). The preceding paragraph assumes that cholesterol loss occurs primarily during capacitation. However, if such loss is delayed until after initiation of the acrosome reaction, a mechanism other than cholesterol resorption by exogenous acceptors is probably involved. In the testis, the activity of cholesteryl ester hydrolases is regulated by protein kinase A and these hydrolases have been implicated in controlling the membrane cholesterol levels (Baily and Grogan, 1986). Since protein kinase A is activated during the human sperm acrosome reaction (see below) and has an important role in this process, it is possible that the same mechanism functions to regulate sperm membrane cholesterol in spermatozoa as in the testis. Concomitant with a decrease in the cholesterol/phospholipid ratio, some evidence suggests that a migration of membrane 1268
proteins and Iipids occurs. Together, these processes are thought to create areas in the plasma membrane that are unstable ('fusogenic') and more permeable. These areas are proposed to represent fusion sites for the OAM and the plasma membrane (see section on Acrosome reaction). Acrosome reaction General considerations Two major problems associated with studying the acrosome reaction have led to much confusion and contradiction. The first is the detection of the acrosome reaction. Currently several different techniques are employed for the human, including the use of antibodies against plasma membrane proteins, chlortetracycline, lectins and differential staining (Cross and Meizel, 1989). Each may detect a different step in the acrosome reaction. The first two are probably primarily an indication of the loss of the plasma membrane (and possibly some of the OAM), whereas the latter two indicate that the acrosome reaction has proceeded to its final stages (the partial or complete loss of acrosomal components). Depending on the staining technique, a different interpretation can be made on whether or not the acrosome reaction has taken place. A second problem is that the acrosome reaction (or at least a process that appears similar to it) can be induced fairly easily by a number of agents that may not represent the actual mechanism. For instance, any chemical that alters the lipid composition of the membrane can potentially induce membrane changes that simulate the early stages of the acrosome reaction. Thus, it is almost impossible to know whether acrosome reaction modulators act pharmacologically or physiologically. For this reason, the regulated pathway of secretion (exocytosis) of certain somatic cells will be used as our model. It is assumed that if processes are found to occur in spermatozoa that mimic those of such secretory cells, they are probably of physiological importance. This is not an unreasonable assumption because the basic components of the exocytotic mechanisms are probably widespread and highly conserved given the essential nature of exocytosis for cell and tissue function. In the regulated pathway of secretion, secretory vesicles migrate to the plasma membrane but make no contact with this membrane unless the appropriate signal is received. Cytoskeletal and osmotic forces retain the vesicles in place until that time. It is likely that the same forces are present in spermatozoa so that the spatial arrangement of the plasma membrane and acrosome is maintained during sperm transport. In order for fusion of the OAM and plasma membrane to occur, these forces have to be overcome. It should be kept in mind that the acrosome reaction, which is essential for fertilization and, ultimately, for the propagation of the species, probably does not depend on a single biochemial mechanism. Primary and secondary mechanisms are most probably present whereby the acrosome reaction can occur. Furthermore, species variations may have arisen during the process of evolution. External signals (ligands) The signal(s) of the initiation of the acrosome reaction are most likely received by one or more receptors on the plasma membrane
Human sperm capacitation and the acrosome reaction
surface, which transmit them across the plasma membrane. The physiological signal(s) for the acrosome reaction remain(s) to be established. A zona glycoprotein (ZP3) can induce the acrosome reaction and may represent the signal occurring after the spermatozoon binds to the zona pellucida. However earlier signals, such as progesterone, prostaglandins, sterol sulphatase, glycosaminoglycans and/or yet unidentified factors, are present in follicular fluid and cumulus cell secretions and can induce the acrosome reaction. Progesterone (e.g. Blackmore et al., 1990) and prostaglandins may act by promoting Ca 2+ transport. Ion transport As with somatic cell exocytosis, the acrosome reaction can be induced by conditions which cause a sharp rise in intracellular Ca 2+ and the first step in the acrosome reaction is often said to be a large influx of calcium ions. In rodent spermatozoa, Ca 2+ influx appears to be capacitation sensitive. For instance, when noncapacitated guinea-pig spermatozoa are placed in a medium containing high Ca 2+ concentrations, no acrosome reaction occurs, whereas this reaction takes place when capacitated spermatozoa are placed in the same medium. Apparently Ca2 + transport into the cell is blocked in noncapacitated spermatozoa or the Ca 2+ ions are rapidly removed after entry. Human spermatozoa act somewhat differently. Even after capacitation, exposure of these spermatozoa to media containing high Ca 2+ concentrations does not induce the acrosome reaction. However, both capacitated and noncapacitated human spermatozoa can respond to Ca2+-containing media in the presence of a calcium ionophore (renders the plasma membrane permeable to Ca2+ or releases Ca 2+ from internal stores). This may mean that although the influx of Ca 2+ can induce the human sperm acrosome reaction, it is not the initiating factor. A large Ca 2+ influx may not even be essential for the human acrosome reaction because stimulation of certain G-proteins (Anderson et al., 1991) or protein kinase A (De Jonge et al., 1991b) induces the acrosome reaction in the nominal absence of Ca 2+ from the medium. Besides Ca2 + , it is likely that changes also occur in the transport of other ions such as Na + , K+ and H + . Ion transport is primarily controlled by enzymes and channel proteins because the plasma membrane is only poorly soluble to charged ions. A rapid influx of Ca 2+ usually occurs in somatic cells by the opening of calcium channels but little information is available in this regard for spermatozoa. However, ion transport enzymes are known to be associated with spermatozoa. Two of these enzymes are Na+K + -ATPase and Ca2 + -ATPase which, respectively, pump Na + and Ca2"1" out of the cell. The Na + K + -ATPase maintains osmotic balance and cell volume by maintaining ion concentrations. A proton pump, possibly Mg2+-ATPase, is also present in spermatozoa and may maintain high intra-acrosomal H + levels (the acrosomal pH is
OPINION
Human sperm capacitation and the acrosome reaction*
L.J.D.Zaneveld1, C.J.De Jonge, R.A.Anderson and S.R.Mack
Key words: spermatozoa/acrosome/human/G-proteins/protein phosphorylation
Departments of Obstetrics and Gynecology, Biochemistry and Physiology, Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA
Introduction
'To whom correspondence should be addressed
A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phosphoupid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion. •Presented at the 2nd Dusseldorf Symposium on Interactions in Reproductive Medicine, November 18-20, 1990. Prepared for publication by N.J.Alexander, G.Freundl and V.Insler.
© Oxford University Press
The requirement for epididymal or ejaculated spermatozoa to undergo modifications before they are able to fertilize the oocyte was first described in 1951. Numerous articles have addressed the biochemical, physiological and morphological aspects of capacitation and the acrosome reaction, as summarized in a variety of reviews (for example, McRorie and Williams, 1974; Hoskins and Casillas, 1975; Bedford and Cooper, 1978; Stambaugh, 1978; Meizel, 1978, 1984, 1985; Green, 1978; O'Rand, 1979; Bhattacharyya and Zaneveld, 1982; Rogers and Bentwood, 1982; Clegg, 1983; Monroy and Rosati, 1983; Chang, 1984; Hinrichsen-Kohane et ai, 1984; Austin, 1985; Langlais and Roberts, 1985; Tesarik, 1986; Peterson etal., 1987; Wasserman, 1987; Yanagimachi, 1988; Kopf, 1988, Garbers, 1989; Garbers and Kopf, 1989; Shur, 1989; Saling, 1991; Zaneveld and De Jonge, 1991). In spite of the extensive literature, there is almost as much confusion on the subject now as there was two decades ago. This confusion is caused by the many factors and conditions that can influence capacitation and/or the acrosome reaction under artificial conditions and the uncertainty as to which are physiological. The observations often appear to be unrelated to each other so that it has been difficult to combine the data into a unifying hypothesis. In recent years, however, much knowledge has accrued regarding the functional activity of membranes in somatic cells. It is likely that this knowledge also can be applied to spermatozoa. The following discussion is based on four hypotheses: (i) One or more of the lytic enzymes of the sperm acrosome has an essential role in the penetration of the spermatozoon through one or more of the layers surrounding the oocyte. (ii) Through the processes of capacitation and the acrosome reaction, the activation/release of the lytic enzyme(s) is controlled so that it does not occur until the spermatozoon reaches the target layer(s) of the oocyte. (iii) At least part of capacitation involves the biochemical modification of the sperm head membrane(s), allowing the acrosome reaction to occur with the appropriate oocyte signal(s). (iv) The acrosome reaction is a type of exocytosis. Due to limitations of space, only some of the more recent references that cannot be found in the reviews (see above), are cited. This review focuses on the human. 1265
L.J.D.Zaneveld et al.
Physiological importance of capacitation and the acrosome reaction The function of the spermatozoon is to deliver DNA into the oocyte. In order for this to happen, spermatozoa first pass through the female genital tract and then through the various oocyte investments, namely the cumulus oophorus, the corona radiata, the zona pellucida and the vitelline membrane (oolemma). The cumulus oophorus and the corona radiata are layers of follicle (granulosa) cells. The cumulus cell matrix consists mostly of hyaluronic acid. The zona pellucida consists of glycoproteins, the vitelline membrane is the plasma membrane that surrounds the oocyte. The fertilizing spermatozoon enters the oocyte while these layers are intact. To do so, the spermatozoon uses its forward progressive motion and lytic enzymes. The latter digest a hole through their target layers in front of the spermatozoon. As their function necessitates, the enzymes are associated with the most anterior portion of the sperm head: the sperm acrosome. The acrosome is a flattened, membrane-bound vesicle that surrounds the anterior portion of the sperm nucleus. Because of its flattened appearance, the acrosome can be divided into three parts: (i) the inner acrosomal membrane (IAM) that overlies the nuclear membrane; (ii) the outer acrosomal membrane (OAM) which represents the outer layer of the vesicle; and (iii) the acrosome proper, located between the IAM and the OAM. The IAM and OAM join at the equatorial segment. The entire spermatozoon, including the acrosome, is surrounded by the plasma membrane. A small area of cytosol is present between the OAM and plasma membrane (cytosolic space). The area inside the acrosome is referred to as the intra-acrosomal space. The acrosome may have similarities to a secretory vesicle that contains digestive enzymes which are released through the process of exocytosis when the cell receives the appropriate signal ('regulated pathway of secretion'). The most intensively investigated acrosomal enzyme is acrosin, a serine proteinase. Numerous studies have indicated that acrosin is essential for fertilization. The enzyme appears to have a role in sperm binding to the zona pellucida, sperm penetration (lysis) of the zona pellucida and the acrosome reaction (see below). Acrosin is almost entirely present in an inactive form (proacrosin). Proacrosin is primarily associated within the acrosome proper and IAM although some proacrosin can be found on the plasma membrane. It can be speculated that surface proacrosin is involved in the binding of the spermatozoon to the zona pellucida, whereas the intra-acrosomal proacrosin is involved in the acrosome reaction and zona lysis. Intra-acrosomal proacrosin is converted to acrosin and exposed by the partial or complete removal of the plasma membrane and OAM, before acrosin can exert its activity. This is probably the reason why morphologically unmodified (non-acrosome reacted) spermatozoa cannot penetrate the zona pellucida. The following model utilizes acrosin as an example but the same model probably applies to other sperm-head enzymes. For the correct utilization of the enzyme and to ensure that this highly lytic enzyme does not damage non-target tissue, activation of proacrosin and release of acrosin must be coordinated with sperm approximation to the oocyte. We propose that such selective exposure is controlled by sperm capacitation 1266
and the acrosome reaction. Capacitation can be defined as the biochemical modifications that are required for the acrosome reaction to occur in response to the appropriate stimulus. The acrosome reaction involves the localized fusion of certain sections of the plasma membrane with the OAM, the lysis/disappearance of these fused areas, the formation of vesicles by the remaining PM and OAM, the dispersal of these vesicles and the release of the acrosomal contents. After the acrosome reaction, only the IAM remains attached to the spermatozoon. No proacrosin activation occurs during capacitation but as the acrosome reaction takes place, a large proportion of the proacrosin is converted to acrosin which is then released. Physiologically, it would be most advantageous if the acrosome reaction is completed, i.e. if acrosin is released, after the spermatozoon binds to the zona pellucida so that the enzyme exerts its activity specifically at the site where sperm binding takes place. Presently, it is argued whether the acrosome reaction occurs before or after sperm binding to the zona. Acrosomereacted spermatozoa have not only been detected on the zona pellucida but also in the follicle cell layers of the oocyte. Furthermore, the acrosome reaction can be stimulated by both zona glycoproteins and follicle (cumulus) cell secretions. Therefore, it is reasonable to assume that the acrosome reaction can be initiated while the spermatozoon moves through the follicle cell layers. Since such transport is fairly rapid, the acrosome reaction is most probably not completed until after the spermatozoon binds to the zona pellucida. However, in the case that sperm transport through the follicle cell layer is delayed, the acrosome reaction may be completed before the spermatozoon reaches the zona pellucida. Whether such spermatozoa can penetrate the zona pellucida remains to be established but they are probably disadvantaged (e.g. Liu and Baker, 1990). Some proacrosin remains bound to the IAM after the acrosome reaction is completed. Preliminary evidence tends to suggest that the IAM-bound proacrosin is activated as the spermatozoon passes through the zona, thus favouring the formation of the penetration slit in front of the spermatozoon. It also appears that capacitation and the acrosome reaction have to occur before spermatozoa can fuse with the vitelline membrane (the basis of the zona-free hamster oocyte penetration assay). Acrosin and /or other proteolytic enzymes may be involved in vitelline membrane penetration since proteinase inhibitors prevent this process.
Capacitation Location and control It appears that spermatozoa can become capacitated in any of the tubular organs of the female genital tract (except possibly the vagina) and in the cumulus oophorus. However, the efficacy of the sites may vary. Sperm transport through the female genital tract can occur quite rapidly (times as short as 15 —30 min have been reported in the human), whereas capacitation requires a much longer time ( 3 - 4 h is estimated for the human). Thus, it can be speculated that the fertilizing spermatozoon gradually undergoes capacitation as it moves through the female genital tract but does not complete the process until after it has entered the cumulus oophorus. This is physiologically beneficial because
Human sperm capacitation and the acrosome reaction
the spermatozoon remains unresponsive to signals inducing the acrosome reaction until it approaches the zona pellucida, preventing a premature acrosome reaction. However, under conditions of long-term storage, the capacitation process can be initiated and, presumably, even be completed in any one of the tubular genital organs. Spermatozoa only become capacitated in the oestrogen- and not the progesterone-stimulated genital tract. The effect of these steroids is not directly on the spermatozoon and is probably mediated via the genital tract. The capacitation factors in the genital tract secretions remain to be identified although several that act in vitro (see below) are also known to be present in genital tract secretions. Identification of the physiologically active factors is problematic since the incubation of spermatozoa in isolated uterine or Fallopian tube secretions inconsistently induces capacitation. One explanation is that contact with the endometrium or endosalpinx is required but some evidence argues against this possibility. Mechanism General considerations The plasma membrane consists of a lipid bilayer in which numerous proteins are dispersed. As with somatic cells, the predominant lipids of the sperm bilayer are phospholipids, cholesterol and glycolipids. The cholesterol/phospholipid ratio of the human sperm plasma membrane is about unity (Mack et ai, 1986). In somatic cells, the integral membrane proteins often contain carbohydrates (i.e. are glycoproteins) and can extend from the outer (extracellular) surface of the membrane to the inner (cytosolic) surface. These proteins have important functions, including ion transport and receptor activation. The carbohydrate portions of the glycolipids and the glycoproteins are always orientated away from the the cytosolic surface of the membrane, i.e. located on the extracellular surface of the plasma membrane but on the inner surface of secretory vesicles. The carbohydrate-rich, outer surface of the plasma membrane is charged and often called the glycocalyx. It also contains absorbed glycoproteins or polysaccharides which bind via noncovalent interactions and do not extend into the lipid bilayer. Such peripheral membrane components can be removed, for instance, by high or low ionic strength solutions or extreme pH variations. In somatic cells, it is known that these peripheral components can block membrane receptors or the function of transport proteins. Removal of peripheral membrane factors Factors may exist in or on the sperm surface which block the acrosome reaction or prevent sperm binding to the zona (such binding may be required for the completion of the acrosome reaction). Acrosome reaction blockers could conceivably be peripheral membrane factors that prevent extracellular signals from interacting with sperm surface receptors, or inhibit ion channels or enzymes, and/or are intrinsic, structural factors which stabilize or otherwise alter the plasma membrane lipid bilayer so that it cannot respond. Removal of these factors is required before the acrosome reaction can occur and is an essential aspect of the capacitation process. Spermatozoa absorb testicular, epididymal and seminal fluid
components during their transport through the male genital tract and during ejaculation. When incubated in seminal plasma, capacitated spermatozoa lose their ability to undergo the acrosome reaction and to penetrate the oocyte, i.e. become 'decapacitated'. Such decapacitated spermatozoa must be 'recapacitated' before they are capable of fertilizing, thus, an early step in capacitation is probably the removal of certain inhibitory factors. The reversible binding of these factors suggests that they are peripheral membrane components and part of the glycocalyx. Many epididymal and seminal factors adhere to spermatozoa but to date only three are known to prevent fertilization reversibly. One of these is a group of relatively high molecular weight (Mr) glycoproteins (180 to 260 kDa) for which the terms 'decapacitation factor', 'antifertility factor', 'acrosomestabilizing factor' and 'acrosome-reaction inhibiting factor' have been used. It appears that these glycoproteins differ in the mechanism by which they prevent the fertilizing capacity of spermatozoa. For instance, our laboratory has shown diat one high Mr human factor prevents the acrosome reaction, whereas another prevents sperm penetration through the zona pellucida without an apparent effect on the acrosome reaction, possibily by preventing sperm binding. The mechanism of action of these gycoproteins is unknown. Their origin is primarily the seminal plasma although they can also be found in epididymal fluid. Another epididymal/seminal glycoprotein which reversibly prevents capacitation, is acrostatin. Human acrostatin has a M r of ~ 5 kDa and inhibits acrosin. Since proacrosin may be a zona binding protein and since acrosin is involved in die acrosome reaction (see further), acrostatin probably acts by inhibiting either or both of these processes. In the mouse, an epididymal acrosin inhibitor is known to prevent zona binding. The third factor is a high M r polylactosaminyl glycoside which has been isolated from mouse epididymal fluid. This factor inhibits galactosyltransferase, an enzyme on the mouse sperm membrane diat also appears to be involved in zona binding. The presence of this inhibitor in human seminal plasma and a role for galactosyltransferase in zona binding of human spermatozoa remains to be established. Any agent or method that (i) disrupts the noncovalent bonds between the peripheral membrane factors and the sperm surface; (ii) alters the carbohydrate content of the glycocalyx and/or (iii) hydrolyses the factors themselves, may cause the removal or destruction of the inhibitory agents and initiate capacitation. Thus, it is not surprising that a variety of techniques have been reported to induce capacitation in vitro, including treatment of spermatozoa with high salt concentrations, glycosaminoglycans, carbohydrases and proteinases. Albumin, a frequendy used agent for in-vitro capacitation, removes glycoconjugates from the sperm surface (e.g. Focarelli etai, 1990). Whether any of these in-vitro capacitation promoters are actually involved in the capacitation process in vivo remains to be shown. Capacitation is known to be associated with a decrease in net surface charge and a loss of carbohydrates, including sialic acid. Such changes can also be explained by the removal of peripheral glycoproteins or polysaccharides, which can contain sialic acid such as die high M r glycoprotein antifertility factors. Changes in the carbohydrate moiety of the glycocalyx may further explain the alterations in lectin binding that are observed after capacitation. 1267
LJ.D.Zanevdd et al.
Changes in membrane lipid composition If capactitation is truly a reversible process (as is generally considered) it is unlikely to be associated with major sturctural changes of the sperm membrane. In addition, structural changes may lead to a premature acrosome reaction so that such changes should optimally occur just before the acrosome reaction takes place, i.e. after the spermatozoon begins to penetrate the oocyte. Furthermore, there is no good evidence to suggest that structural modifications are required before the acrosome reaction can be stimulated. For instance, treatment of non-capacitated (washed ejaculated) human spermatozoa with calcium ionophore or dibutyryl cyclic AMP (dbc AMP), which bypass the need for surface receptor activation (see section on Acrosome reaction), causes the acrosome reaction (Anderson et al., 1990a). Although a generally held belief is that membrane destabilization and permeabilization take place during capacitation, this may not be the case. It is more likely that such modifications are part of the acrosome reaction or occur during the transition period between capacitation and the acrosome reaction. The cholesterol/phospholipid ratio is important for membrane stability. A decrease in cholesterol or an increase in phospholipids causes destabilization of the membrane and an increase in membrane permeability. Thus, it is not surprising that under invitro conditions, the acrosome reaction is stimulated by removal of cholesterol or the addition of phospholipids and that the addition of cholesterol to spermatozoa can hinder the acrosome reaction by 'hardening' the membrane. This had led to the hypothesis that the removal of cholesterol is an integral aspect of capacitation. Membrane vesicles in seminal plasma can donate cholesterol to the plasma membrane and were suggested to be 'decapacitating factors'. Cholesterol loss from membranes usually occurs via carrier proteins or enzymes. Cholesterol acceptors in blood include lipoproteins, apolipoproteins, albumin, and lecithin: cholesterol acyltransferase (LCAT). One or more of these acceptors can be found in female genital secretions and are present in media used for in-vitro capacitation and fertilization, i.e. media containing heat-inactivated blood serum, follicular fluid, albumin or egg yolk. It remains to be established whether or not the components in these media actually function as cholesterol acceptors, because they may affect the sperm membrane by another mechanism. For instance, albumin was shown to donate phospholipids rather than to remove cholesterol (Davis el al., 1980) and may cause removal of peripheral membrane glycoconjugates (see above). The preceding paragraph assumes that cholesterol loss occurs primarily during capacitation. However, if such loss is delayed until after initiation of the acrosome reaction, a mechanism other than cholesterol resorption by exogenous acceptors is probably involved. In the testis, the activity of cholesteryl ester hydrolases is regulated by protein kinase A and these hydrolases have been implicated in controlling the membrane cholesterol levels (Baily and Grogan, 1986). Since protein kinase A is activated during the human sperm acrosome reaction (see below) and has an important role in this process, it is possible that the same mechanism functions to regulate sperm membrane cholesterol in spermatozoa as in the testis. Concomitant with a decrease in the cholesterol/phospholipid ratio, some evidence suggests that a migration of membrane 1268
proteins and Iipids occurs. Together, these processes are thought to create areas in the plasma membrane that are unstable ('fusogenic') and more permeable. These areas are proposed to represent fusion sites for the OAM and the plasma membrane (see section on Acrosome reaction). Acrosome reaction General considerations Two major problems associated with studying the acrosome reaction have led to much confusion and contradiction. The first is the detection of the acrosome reaction. Currently several different techniques are employed for the human, including the use of antibodies against plasma membrane proteins, chlortetracycline, lectins and differential staining (Cross and Meizel, 1989). Each may detect a different step in the acrosome reaction. The first two are probably primarily an indication of the loss of the plasma membrane (and possibly some of the OAM), whereas the latter two indicate that the acrosome reaction has proceeded to its final stages (the partial or complete loss of acrosomal components). Depending on the staining technique, a different interpretation can be made on whether or not the acrosome reaction has taken place. A second problem is that the acrosome reaction (or at least a process that appears similar to it) can be induced fairly easily by a number of agents that may not represent the actual mechanism. For instance, any chemical that alters the lipid composition of the membrane can potentially induce membrane changes that simulate the early stages of the acrosome reaction. Thus, it is almost impossible to know whether acrosome reaction modulators act pharmacologically or physiologically. For this reason, the regulated pathway of secretion (exocytosis) of certain somatic cells will be used as our model. It is assumed that if processes are found to occur in spermatozoa that mimic those of such secretory cells, they are probably of physiological importance. This is not an unreasonable assumption because the basic components of the exocytotic mechanisms are probably widespread and highly conserved given the essential nature of exocytosis for cell and tissue function. In the regulated pathway of secretion, secretory vesicles migrate to the plasma membrane but make no contact with this membrane unless the appropriate signal is received. Cytoskeletal and osmotic forces retain the vesicles in place until that time. It is likely that the same forces are present in spermatozoa so that the spatial arrangement of the plasma membrane and acrosome is maintained during sperm transport. In order for fusion of the OAM and plasma membrane to occur, these forces have to be overcome. It should be kept in mind that the acrosome reaction, which is essential for fertilization and, ultimately, for the propagation of the species, probably does not depend on a single biochemial mechanism. Primary and secondary mechanisms are most probably present whereby the acrosome reaction can occur. Furthermore, species variations may have arisen during the process of evolution. External signals (ligands) The signal(s) of the initiation of the acrosome reaction are most likely received by one or more receptors on the plasma membrane
Human sperm capacitation and the acrosome reaction
surface, which transmit them across the plasma membrane. The physiological signal(s) for the acrosome reaction remain(s) to be established. A zona glycoprotein (ZP3) can induce the acrosome reaction and may represent the signal occurring after the spermatozoon binds to the zona pellucida. However earlier signals, such as progesterone, prostaglandins, sterol sulphatase, glycosaminoglycans and/or yet unidentified factors, are present in follicular fluid and cumulus cell secretions and can induce the acrosome reaction. Progesterone (e.g. Blackmore et al., 1990) and prostaglandins may act by promoting Ca 2+ transport. Ion transport As with somatic cell exocytosis, the acrosome reaction can be induced by conditions which cause a sharp rise in intracellular Ca 2+ and the first step in the acrosome reaction is often said to be a large influx of calcium ions. In rodent spermatozoa, Ca 2+ influx appears to be capacitation sensitive. For instance, when noncapacitated guinea-pig spermatozoa are placed in a medium containing high Ca 2+ concentrations, no acrosome reaction occurs, whereas this reaction takes place when capacitated spermatozoa are placed in the same medium. Apparently Ca2 + transport into the cell is blocked in noncapacitated spermatozoa or the Ca 2+ ions are rapidly removed after entry. Human spermatozoa act somewhat differently. Even after capacitation, exposure of these spermatozoa to media containing high Ca 2+ concentrations does not induce the acrosome reaction. However, both capacitated and noncapacitated human spermatozoa can respond to Ca2+-containing media in the presence of a calcium ionophore (renders the plasma membrane permeable to Ca2+ or releases Ca 2+ from internal stores). This may mean that although the influx of Ca 2+ can induce the human sperm acrosome reaction, it is not the initiating factor. A large Ca 2+ influx may not even be essential for the human acrosome reaction because stimulation of certain G-proteins (Anderson et al., 1991) or protein kinase A (De Jonge et al., 1991b) induces the acrosome reaction in the nominal absence of Ca 2+ from the medium. Besides Ca2 + , it is likely that changes also occur in the transport of other ions such as Na + , K+ and H + . Ion transport is primarily controlled by enzymes and channel proteins because the plasma membrane is only poorly soluble to charged ions. A rapid influx of Ca 2+ usually occurs in somatic cells by the opening of calcium channels but little information is available in this regard for spermatozoa. However, ion transport enzymes are known to be associated with spermatozoa. Two of these enzymes are Na+K + -ATPase and Ca2 + -ATPase which, respectively, pump Na + and Ca2"1" out of the cell. The Na + K + -ATPase maintains osmotic balance and cell volume by maintaining ion concentrations. A proton pump, possibly Mg2+-ATPase, is also present in spermatozoa and may maintain high intra-acrosomal H + levels (the acrosomal pH is
