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 Key words: spermatozoa/acrosome/human/G-proteins/protein

S.R.Mack 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
The requirement for epididymal or ejaculated spermatozoa to
A model is presented that describes the mechanism of human undergo modifications before they are able to fertilize the oocyte
sperm capacitation and the acrosome reaction. The processes was first described in 1951. Numerous articles have addressed
of capacitation and the acrosome reaction are proposed to the biochemical, physiological and morphological aspects of
function in control of the activation/release of acrosomal capacitation and the acrosome reaction, as summarized in a
enzyme(s) involved in sperm penetration through the zona variety of reviews (for example, McRorie and Williams, 1974;
pellucida. During capacitation, the sperm head membranes Hoskins and Casillas, 1975; Bedford and Cooper, 1978;
are biochemically modified, allowing the acrosome reaction Stambaugh, 1978; Meizel, 1978, 1984, 1985; Green, 1978;
to take place when the spermatozoon approaches or reaches O'Rand, 1979; Bhattacharyya and Zaneveld, 1982; Rogers and
the zona pellucida, resulting in the localized activation and Bentwood, 1982; Clegg, 1983; Monroy and Rosati, 1983; Chang,
release of the appropriate enzyme(s). Further, capacitation 1984; Hinrichsen-Kohane et ai, 1984; Austin, 1985; Langlais
is presented as a continuing process that occurs during sperm and Roberts, 1985; Tesarik, 1986; Peterson etal., 1987;
transport through the female genital tract and is Wasserman, 1987; Yanagimachi, 1988; Kopf, 1988, Garbers,
physiologically not completed until the spermatozoon reaches 1989; Garbers and Kopf, 1989; Shur, 1989; Saling, 1991;
the oocyte (unless the spermatozoa are kept at a particular Zaneveld and De Jonge, 1991). In spite of the extensive literature,
genital tract site for prolonged periods). The biochemical there is almost as much confusion on the subject now as there
alterations that occur during capacitation are discussed. It was two decades ago. This confusion is caused by the many
is suggested that extensive modifications in the lipid bilayer factors and conditions that can influence capacitation and/or the
structure, e.g. in the cholesterol or phosphoupid content, are acrosome reaction under artificial conditions and the uncertain-
not part of capacitation because such changes would ty as to which are physiological. The observations often appear
prematurely destabilize the membranes. Rather, such changes to be unrelated to each other so that it has been difficult to com-
occur during the acrosome reaction. It is also proposed that bine the data into a unifying hypothesis. In recent years, however,
the human sperm acrosome reaction has many similarities much knowledge has accrued regarding the functional activity
to the somatic cell exocytotic events which occur during the of membranes in somatic cells. It is likely that this knowledge
regulated pathway of secretion. One or more oocyte stimuli also can be applied to spermatozoa.
result in the activation of protein kinases, likely (but not The following discussion is based on four hypotheses:
necessarily) via activation of G-protein coupled receptors on (i) One or more of the lytic enzymes of the sperm acrosome has
the sperm plasma membrane and the formation of second an essential role in the penetration of the spermatozoon through
messengers. The kinases phosphorylate and activate proteins, one or more of the layers surrounding the oocyte.
continuing the biochemical cascade that ultimately results in (ii) Through the processes of capacitation and the acrosome
the acrosome reaction. The role of other enzyme systems such reaction, the activation/release of the lytic enzyme(s) is controlled
as those involved in ion transport, proteolysis, phospholipid so that it does not occur until the spermatozoon reaches the target
metabolism (including that of arachidonic acid) and other layer(s) of the oocyte.
metabolic events, is discussed. Calcium ion influx as initiator (iii) At least part of capacitation involves the biochemical
of the acrosome reaction is reconsidered. The proposed model modification of the sperm head membrane(s), allowing the
also takes into consideration the structural events of mem- acrosome reaction to occur with the appropriate oocyte signal(s).
brane fusion. (iv) The acrosome reaction is a type of exocytosis.
•Presented at the 2nd Dusseldorf Symposium on Interactions in Due to limitations of space, only some of the more recent
Reproductive Medicine, November 18-20, 1990. Prepared for references that cannot be found in the reviews (see above), are
publication by N.J.Alexander, G.Freundl and V.Insler. cited. This review focuses on the human.

© Oxford University Press 1265

L.J.D.Zaneveld et al.

Physiological importance of capacitation and the and the acrosome reaction. Capacitation can be defined as the
acrosome reaction biochemical modifications that are required for the acrosome
reaction to occur in response to the appropriate stimulus. The
The function of the spermatozoon is to deliver DNA into the acrosome reaction involves the localized fusion of certain sec-
oocyte. In order for this to happen, spermatozoa first pass through tions of the plasma membrane with the OAM, the lysis/dis-
the female genital tract and then through the various oocyte appearance of these fused areas, the formation of vesicles by the
investments, namely the cumulus oophorus, the corona radiata, remaining PM and OAM, the dispersal of these vesicles and the
the zona pellucida and the vitelline membrane (oolemma). The release of the acrosomal contents. After the acrosome reaction,
cumulus oophorus and the corona radiata are layers of follicle only the IAM remains attached to the spermatozoon. No pro-
(granulosa) cells. The cumulus cell matrix consists mostly of acrosin activation occurs during capacitation but as the acrosome
hyaluronic acid. The zona pellucida consists of glycoproteins, reaction takes place, a large proportion of the proacrosin is con-
the vitelline membrane is the plasma membrane that surrounds verted to acrosin which is then released.
the oocyte. The fertilizing spermatozoon enters the oocyte while Physiologically, it would be most advantageous if the acrosome
these layers are intact. To do so, the spermatozoon uses its reaction is completed, i.e. if acrosin is released, after the
forward progressive motion and lytic enzymes. The latter digest spermatozoon binds to the zona pellucida so that the enzyme
a hole through their target layers in front of the spermatozoon. exerts its activity specifically at the site where sperm binding takes
As their function necessitates, the enzymes are associated with place. Presently, it is argued whether the acrosome reaction
the most anterior portion of the sperm head: the sperm acrosome. occurs before or after sperm binding to the zona. Acrosome-
The acrosome is a flattened, membrane-bound vesicle that reacted spermatozoa have not only been detected on the zona
surrounds the anterior portion of the sperm nucleus. Because of pellucida but also in the follicle cell layers of the oocyte.
its flattened appearance, the acrosome can be divided into three Furthermore, the acrosome reaction can be stimulated by both
parts: (i) the inner acrosomal membrane (IAM) that overlies the zona glycoproteins and follicle (cumulus) cell secretions.
nuclear membrane; (ii) the outer acrosomal membrane (OAM) Therefore, it is reasonable to assume that the acrosome reaction
which represents the outer layer of the vesicle; and (iii) the can be initiated while the spermatozoon moves through the follicle
acrosome proper, located between the IAM and the OAM. The cell layers. Since such transport is fairly rapid, the acrosome
IAM and OAM join at the equatorial segment. The entire reaction is most probably not completed until after the
spermatozoon, including the acrosome, is surrounded by the spermatozoon binds to the zona pellucida. However, in the case
plasma membrane. A small area of cytosol is present between that sperm transport through the follicle cell layer is delayed,
the OAM and plasma membrane (cytosolic space). The area the acrosome reaction may be completed before the spermatozoon
inside the acrosome is referred to as the intra-acrosomal space. reaches the zona pellucida. Whether such spermatozoa can
The acrosome may have similarities to a secretory vesicle that penetrate the zona pellucida remains to be established but they
contains digestive enzymes which are released through the process are probably disadvantaged (e.g. Liu and Baker, 1990).
of exocytosis when the cell receives the appropriate signal Some proacrosin remains bound to the IAM after the acrosome
('regulated pathway of secretion'). reaction is completed. Preliminary evidence tends to suggest that
The most intensively investigated acrosomal enzyme is acrosin, the IAM-bound proacrosin is activated as the spermatozoon passes
a serine proteinase. Numerous studies have indicated that acrosin through the zona, thus favouring the formation of the penetration
is essential for fertilization. The enzyme appears to have a role slit in front of the spermatozoon. It also appears that capacitation
in sperm binding to the zona pellucida, sperm penetration (lysis) and the acrosome reaction have to occur before spermatozoa can
of the zona pellucida and the acrosome reaction (see below). fuse with the vitelline membrane (the basis of the zona-free
Acrosin is almost entirely present in an inactive form hamster oocyte penetration assay). Acrosin and /or other
(proacrosin). Proacrosin is primarily associated within the proteolytic enzymes may be involved in vitelline membrane
acrosome proper and IAM although some proacrosin can be penetration since proteinase inhibitors prevent this process.
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 Capacitation
involved in the acrosome reaction and zona lysis. Intra-acrosomal
proacrosin is converted to acrosin and exposed by the partial or Location and control
complete removal of the plasma membrane and OAM, before It appears that spermatozoa can become capacitated in any of
acrosin can exert its activity. This is probably the reason why the tubular organs of the female genital tract (except possibly
morphologically unmodified (non-acrosome reacted) spermatozoa the vagina) and in the cumulus oophorus. However, the efficacy
cannot penetrate the zona pellucida. The following model utilizes of the sites may vary. Sperm transport through the female genital
acrosin as an example but the same model probably applies to tract can occur quite rapidly (times as short as 15 —30 min have
other sperm-head enzymes. been reported in the human), whereas capacitation requires a
For the correct utilization of the enzyme and to ensure that much longer time ( 3 - 4 h is estimated for the human). Thus,
this highly lytic enzyme does not damage non-target tissue, it can be speculated that the fertilizing spermatozoon gradually
activation of proacrosin and release of acrosin must be undergoes capacitation as it moves through the female genital
coordinated with sperm approximation to the oocyte. We propose tract but does not complete the process until after it has entered
that such selective exposure is controlled by sperm capacitation the cumulus oophorus. This is physiologically beneficial because

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the spermatozoon remains unresponsive to signals inducing the components during their transport through the male genital tract
acrosome reaction until it approaches the zona pellucida, and during ejaculation. When incubated in seminal plasma,
preventing a premature acrosome reaction. However, under capacitated spermatozoa lose their ability to undergo the acrosome
conditions of long-term storage, the capacitation process can be reaction and to penetrate the oocyte, i.e. become 'decapacitated'.
initiated and, presumably, even be completed in any one of the Such decapacitated spermatozoa must be 'recapacitated' before
tubular genital organs. they are capable of fertilizing, thus, an early step in capacitation
Spermatozoa only become capacitated in the oestrogen- and is probably the removal of certain inhibitory factors. The
not the progesterone-stimulated genital tract. The effect of these reversible binding of these factors suggests that they are peripheral
steroids is not directly on the spermatozoon and is probably membrane components and part of the glycocalyx.
mediated via the genital tract. The capacitation factors in the Many epididymal and seminal factors adhere to spermatozoa
genital tract secretions remain to be identified although several but to date only three are known to prevent fertilization reversibly.
that act in vitro (see below) are also known to be present in genital One of these is a group of relatively high molecular weight (Mr)
tract secretions. Identification of the physiologically active factors glycoproteins (180 to 260 kDa) for which the terms
is problematic since the incubation of spermatozoa in isolated 'decapacitation factor', 'antifertility factor', 'acrosomestabilizing
uterine or Fallopian tube secretions inconsistently induces factor' and 'acrosome-reaction inhibiting factor' have been used.
capacitation. One explanation is that contact with the endometrium It appears that these glycoproteins differ in the mechanism by
or endosalpinx is required but some evidence argues against this which they prevent the fertilizing capacity of spermatozoa. For
possibility. instance, our laboratory has shown diat one high Mr human
factor prevents the acrosome reaction, whereas another prevents
Mechanism sperm penetration through the zona pellucida without an apparent
General considerations effect on the acrosome reaction, possibily by preventing sperm
The plasma membrane consists of a lipid bilayer in which binding. The mechanism of action of these gycoproteins is
numerous proteins are dispersed. As with somatic cells, the unknown. Their origin is primarily the seminal plasma although
predominant lipids of the sperm bilayer are phospholipids, they can also be found in epididymal fluid.
cholesterol and glycolipids. The cholesterol/phospholipid ratio Another epididymal/seminal glycoprotein which reversibly
of the human sperm plasma membrane is about unity (Mack prevents capacitation, is acrostatin. Human acrostatin has a M r
et ai, 1986). In somatic cells, the integral membrane proteins of ~ 5 kDa and inhibits acrosin. Since proacrosin may be a zona
often contain carbohydrates (i.e. are glycoproteins) and can binding protein and since acrosin is involved in die acrosome
extend from the outer (extracellular) surface of the membrane reaction (see further), acrostatin probably acts by inhibiting either
to the inner (cytosolic) surface. These proteins have important or both of these processes. In the mouse, an epididymal acrosin
functions, including ion transport and receptor activation. The inhibitor is known to prevent zona binding.
carbohydrate portions of the glycolipids and the glycoproteins The third factor is a high M r polylactosaminyl glycoside
are always orientated away from the the cytosolic surface of the which has been isolated from mouse epididymal fluid. This factor
membrane, i.e. located on the extracellular surface of the plasma inhibits galactosyltransferase, an enzyme on the mouse sperm
membrane but on the inner surface of secretory vesicles. membrane diat also appears to be involved in zona binding. The
The carbohydrate-rich, outer surface of the plasma membrane presence of this inhibitor in human seminal plasma and a role
is charged and often called the glycocalyx. It also contains for galactosyltransferase in zona binding of human spermatozoa
absorbed glycoproteins or polysaccharides which bind via remains to be established.
noncovalent interactions and do not extend into the lipid bilayer. Any agent or method that (i) disrupts the noncovalent bonds
Such peripheral membrane components can be removed, for between the peripheral membrane factors and the sperm surface;
instance, by high or low ionic strength solutions or extreme pH (ii) alters the carbohydrate content of the glycocalyx and/or (iii)
variations. In somatic cells, it is known that these peripheral hydrolyses the factors themselves, may cause the removal or
components can block membrane receptors or the function of destruction of the inhibitory agents and initiate capacitation. Thus,
transport proteins. it is not surprising that a variety of techniques have been reported
to induce capacitation in vitro, including treatment of spermatozoa
Removal of peripheral membrane factors with high salt concentrations, glycosaminoglycans, carbohydrases
Factors may exist in or on the sperm surface which block the and proteinases. Albumin, a frequendy used agent for in-vitro
acrosome reaction or prevent sperm binding to the zona (such capacitation, removes glycoconjugates from the sperm surface
binding may be required for the completion of the acrosome (e.g. Focarelli etai, 1990). Whether any of these in-vitro
reaction). Acrosome reaction blockers could conceivably be capacitation promoters are actually involved in the capacitation
peripheral membrane factors that prevent extracellular signals process in vivo remains to be shown. Capacitation is known to
from interacting with sperm surface receptors, or inhibit ion be associated with a decrease in net surface charge and a loss
channels or enzymes, and/or are intrinsic, structural factors which of carbohydrates, including sialic acid. Such changes can also
stabilize or otherwise alter the plasma membrane lipid bilayer be explained by the removal of peripheral glycoproteins or
so that it cannot respond. Removal of these factors is required polysaccharides, which can contain sialic acid such as die high
before the acrosome reaction can occur and is an essential aspect M r glycoprotein antifertility factors. Changes in the carbo-
of the capacitation process. hydrate moiety of the glycocalyx may further explain the altera-
Spermatozoa absorb testicular, epididymal and seminal fluid tions in lectin binding that are observed after capacitation.

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Changes in membrane lipid composition proteins and Iipids occurs. Together, these processes are thought
If capactitation is truly a reversible process (as is generally to create areas in the plasma membrane that are unstable
considered) it is unlikely to be associated with major sturctural ('fusogenic') and more permeable. These areas are proposed to
changes of the sperm membrane. In addition, structural changes represent fusion sites for the OAM and the plasma membrane
may lead to a premature acrosome reaction so that such changes (see section on Acrosome reaction).
should optimally occur just before the acrosome reaction takes
place, i.e. after the spermatozoon begins to penetrate the oocyte. Acrosome reaction
Furthermore, there is no good evidence to suggest that struc-
tural modifications are required before the acrosome reaction can General considerations
be stimulated. For instance, treatment of non-capacitated (washed Two major problems associated with studying the acrosome
ejaculated) human spermatozoa with calcium ionophore or reaction have led to much confusion and contradiction. The first
dibutyryl cyclic AMP (dbc AMP), which bypass the need for is the detection of the acrosome reaction. Currently several
surface receptor activation (see section on Acrosome reaction), different techniques are employed for the human, including the
causes the acrosome reaction (Anderson et al., 1990a). Although use of antibodies against plasma membrane proteins,
a generally held belief is that membrane destabilization and chlortetracycline, lectins and differential staining (Cross and
permeabilization take place during capacitation, this may not be Meizel, 1989). Each may detect a different step in the acrosome
the case. It is more likely that such modifications are part of the reaction. The first two are probably primarily an indication of
acrosome reaction or occur during the transition period between the loss of the plasma membrane (and possibly some of the
capacitation and the acrosome reaction. OAM), whereas the latter two indicate that the acrosome reaction
The cholesterol/phospholipid ratio is important for membrane has proceeded to its final stages (the partial or complete loss of
stability. A decrease in cholesterol or an increase in phospho- acrosomal components). Depending on the staining technique,
lipids causes destabilization of the membrane and an increase in a different interpretation can be made on whether or not the
membrane permeability. Thus, it is not surprising that under in- acrosome reaction has taken place.
vitro conditions, the acrosome reaction is stimulated by A second problem is that the acrosome reaction (or at least
removal of cholesterol or the addition of phospholipids and that a process that appears similar to it) can be induced fairly easily
the addition of cholesterol to spermatozoa can hinder the by a number of agents that may not represent the actual
acrosome reaction by 'hardening' the membrane. This had led mechanism. For instance, any chemical that alters the lipid
to the hypothesis that the removal of cholesterol is an integral composition of the membrane can potentially induce membrane
aspect of capacitation. Membrane vesicles in seminal plasma can changes that simulate the early stages of the acrosome reaction.
donate cholesterol to the plasma membrane and were suggested Thus, it is almost impossible to know whether acrosome reaction
to be 'decapacitating factors'. modulators act pharmacologically or physiologically. For this
Cholesterol loss from membranes usually occurs via carrier reason, the regulated pathway of secretion (exocytosis) of certain
proteins or enzymes. Cholesterol acceptors in blood include somatic cells will be used as our model. It is assumed that if
lipoproteins, apolipoproteins, albumin, and lecithin: cholesterol processes are found to occur in spermatozoa that mimic those
acyltransferase (LCAT). One or more of these acceptors can be of such secretory cells, they are probably of physiological
found in female genital secretions and are present in media used importance. This is not an unreasonable assumption because the
for in-vitro capacitation and fertilization, i.e. media containing basic components of the exocytotic mechanisms are probably
heat-inactivated blood serum, follicular fluid, albumin or egg widespread and highly conserved given the essential nature of
yolk. It remains to be established whether or not the components exocytosis for cell and tissue function.
in these media actually function as cholesterol acceptors, because In the regulated pathway of secretion, secretory vesicles
they may affect the sperm membrane by another mechanism. For migrate to the plasma membrane but make no contact with this
instance, albumin was shown to donate phospholipids rather than membrane unless the appropriate signal is received. Cytoskeletal
to remove cholesterol (Davis el al., 1980) and may cause removal and osmotic forces retain the vesicles in place until that time.
of peripheral membrane glycoconjugates (see above). It is likely that the same forces are present in spermatozoa so
The preceding paragraph assumes that cholesterol loss occurs that the spatial arrangement of the plasma membrane and
primarily during capacitation. However, if such loss is delayed acrosome is maintained during sperm transport. In order for
until after initiation of the acrosome reaction, a mechanism other fusion of the OAM and plasma membrane to occur, these forces
than cholesterol resorption by exogenous acceptors is probably have to be overcome. It should be kept in mind that the acrosome
involved. In the testis, the activity of cholesteryl ester hydrolases reaction, which is essential for fertilization and, ultimately, for
is regulated by protein kinase A and these hydrolases have been the propagation of the species, probably does not depend on a
implicated in controlling the membrane cholesterol levels (Baily single biochemial mechanism. Primary and secondary
and Grogan, 1986). Since protein kinase A is activated during mechanisms are most probably present whereby the acrosome
the human sperm acrosome reaction (see below) and has an reaction can occur. Furthermore, species variations may have
important role in this process, it is possible that the same arisen during the process of evolution.
mechanism functions to regulate sperm membrane cholesterol
in spermatozoa as in the testis. External signals (ligands)
Concomitant with a decrease in the cholesterol/phospholipid The signal(s) of the initiation of the acrosome reaction are most
ratio, some evidence suggests that a migration of membrane likely received by one or more receptors on the plasma membrane
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 Human sperm capacitation and the acrosome reaction

surface, which transmit them across the plasma membrane. The established. Physiologically, it is possible that these pumps are
physiological signal(s) for the acrosome reaction remain(s) to be either overwhelmed or inhibited during the early stages of the
established. A zona glycoprotein (ZP3) can induce the acrosome acrosome reaction, leading to an influx of Ca 2+ and Na + and
reaction and may represent the signal occurring after the a decrease in intra-acrosomal H + . Changes in these and other
spermatozoon binds to the zona pellucida. However earlier ions may alter the osmotic pressure of the sperm head
signals, such as progesterone, prostaglandins, sterol sulphatase, compartments and cause a rise in acrosomal pH. The rise in pH
glycosaminoglycans and/or yet unidentified factors, are present may cause the activation of enzymes, for instance that of
in follicular fluid and cumulus cell secretions and can induce the proacrosin to acrosin. Inhibition of the ATPases would also
acrosome reaction. Progesterone (e.g. Blackmore et al., 1990) decrease ATP utilization. ATP is the substrate for adenylate
and prostaglandins may act by promoting Ca 2+ transport. cyclase, an enzyme that appears to be regulatory in the acrosome
reaction (see below).
Ion transport The mechansim whereby high cytosolic Ca 2+ can induce the
As with somatic cell exocytosis, the acrosome reaction can be acrosome reaction or exocytosis in somatic cells is poorly
induced by conditions which cause a sharp rise in intracellular understood. In the simplest fusion model (phospholipid vesicles
Ca 2+ and the first step in the acrosome reaction is often said to and a planar phospholipid membrane), Ca 2+ can overcome the
be a large influx of calcium ions. In rodent spermatozoa, Ca 2+ repulsive forces (electrostatic and hydration barriers) between
influx appears to be capacitation sensitive. For instance, when the two membranes by interacting with the polar phospholipid
noncapacitated guinea-pig spermatozoa are placed in a medium head groups (Niles and Cohen, 1991). This allows adhesion to
containing high Ca 2+ concentrations, no acrosome reaction occur but does not lead to fusion unless the osmotic pressure in
occurs, whereas this reaction takes place when capacitated the vesicle is increased, causing swelling of the vesicle. Since
spermatozoa are placed in the same medium. Apparently Ca2 + the acrosome reaction occurs with an increase in cytosolic Ca 2+ ,
transport into the cell is blocked in noncapacitated spermatozoa possibly coupled with an increase in intra-acrosomal osmotic
or the Ca 2+ ions are rapidly removed after entry. pressure and swelling of the acrosome, it is conceivable that this
Human spermatozoa act somewhat differently. Even after simple mechanism is sufficient to explain the acrosome reaction.
capacitation, exposure of these spermatozoa to media containing However, it is generally thought that, at least in somatic cells,
high Ca 2+ concentrations does not induce the acrosome the effects of Ca 2+ are less direct. Ca 2+ may act as a second
reaction. However, both capacitated and noncapacitated human messenger and activate certain enzyme systems. For instance,
spermatozoa can respond to Ca2+-containing media in the phosphoinositide breakdown and protein kinase C activity (see
presence of a calcium ionophore (renders the plasma membrane below) require Ca 2+ (e.g. Roldan and Harrison, 1989). Ca 2+
permeable to Ca2+ or releases Ca 2+ from internal stores). This may also complex with calmodulin which, in turn, can activate
may mean that although the influx of Ca 2+ can induce the Ca2+-calmodulin protein kinase which stimulates protein
phosphorylation. Calmodulin binding to and activation of
human sperm acrosome reaction, it is not the initiating factor.
proacrosin have been reported (Frenette et al., 1990). Finally,
A large Ca 2+ influx may not even be essential for the human
Ca 2+ may directly activate proteins that cause the fusion of the
acrosome reaction because stimulation of certain G-proteins
0AM with the plasma membrane and/or aid in overcoming the
(Anderson et al., 1991) or protein kinase A (De Jonge et al.,
cytoskeletal forces that hold the 0AM and plasma membrane in
1991b) induces the acrosome reaction in the nominal absence
place.
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 Second messenger systems and protein phosphorylation
is primarily controlled by enzymes and channel proteins because The mechanism of exocytosis may vary between different somatic
the plasma membrane is only poorly soluble to charged ions. A cell types and several mechanisms may be employed by the same
rapid influx of Ca 2+ usually occurs in somatic cells by the cell. Both Ca2+-dependent and independent mechanisms have
opening of calcium channels but little information is available been found. In certain cells, Ca 2+ merely acts as a cofactor
in this regard for spermatozoa. However, ion transport enzymes which accelerates the exocytotic response. A common mechanism
are known to be associated with spermatozoa. Two of these in these cases is the membrane receptor activation of GTP-binding
enzymes are Na+K + -ATPase and Ca2 + -ATPase which, proteins (G-proteins) which in turn stimulate second messenger
respectively, pump Na + and Ca2"1" out of the cell. The systems and regulate ion transport mechanisms. Mechanisms can
Na + K + -ATPase maintains osmotic balance and cell volume by change with the availability of stimulatory components. For
maintaining ion concentrations. A proton pump, possibly instance, normally both diacylglycerol and Ca 2+ are required
Mg2+-ATPase, is also present in spermatozoa and may maintain for the activation of protein kinase C (in the presence of
high intra-acrosomal H + levels (the acrosomal pH is <5.0, at phospholipid) but high intracellular Ca 2+ levels as a result of
least in rodent spermatozoa). Antiport systems may also be calcium ionophore treatment of the cell are alone sufficient for
present. Different ATPases appear to be associated with the the activation of the enzyme.
plasma membrane and 0AM so that the ionic composition of In somatic cells, a large number of signals are known to act
the cytosolic and intra-acrosomal spaces may vary. via G-protein-coupled receptors including neurotransmittors,
Inhibition of any one of these three ATPases has been shown peptide and glycoprotein hormones and arachidonic acid
to cause the acrosome reaction of rodent spermatozoa. Whether metabolites (prostaglandins, thromboxanes, leukotrienes)
or not this is true for human spermatozoa remains to be (Birnbaumer, 1990). Data in non-human species are beginning
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L.J.D.Zaneveld el at.

to accumulate which suggest that the acrosome reaction is et al., 1990). Furthermore, kinase C activity has been found in
mediated by G-proteins (Kopf, 1988). Certain G-proteins are human spermatozoa (Rotem et al., 1990; unpublished data from
specifically associated with the acrosomal region and several are our laboratory) and PIP2 hydrolysis takes place when human
located only at the acrosomal tip (Garty et al., 1988). G-proteins spermatozoa are treated with acrosome reaction stimulators
may also have a role in the human sperm acrosome reaction. (Thomas and Meizel, 1989). However, species differences may
Cholera toxin (activates Gs which stimulates the adenylate exist; for instance, no protein kinase C was detected in ram
cyclase system) and pertussis toxin (inactivates several G-proteins, spermatozoa (Roldan and Harrison, 1988).
including the G, which exerts an inhibitory effect on the Other second messenger systems and protein kinases may also
adenylate cyclase system) both stimulate the human acrosome be involved in the acrosome reaction, such as cGMP-dependent
reaction (Anderson et al., 1991). Ca 2+ was not required for the kinase and Ca2+-calmodulin kinase but this is mostly speculative
effect of the toxins. However, these data are still controversial at the present time. More evidence is available for a role of
because Lee et al. (1990) reported that the zona-induced tyrosine kinase in the acrosome reaction (Saling, 1991). In
acrosome reaction of human spermatozoa is inhibited by pertussis contrast to the previous kinases, tyrosine kinase may be associated
toxin. Furthermore, coupling of G-proteins with the adenylate with the cell surface and can act as a receptor for external ligands
cyclase system of spermatozoa is not definite (e.g. Hildebrandt (signals). Recent data suggest that the zona-induced acrosome
etai, 1985). reaction of mouse spermatozoa is mediated by tyrosine kinase
One of the functions of G-proteins is the activation of second (Leyton and Saling, 1989). In somatic cells, tyrosine kinase can
messenger systems. Until recently, little information was available also activate phospholipase C so that the phosphoinositide system
on the role of second messenger systems in the acrosome reaction. can be stimulated without requiring the action of G-proteins
However, it now appears that at least two such pathways func- (Figure 2).
tion in this reaction in the human (De Jonge et al., 1991a,b). Based on these observations, it appears that kinase activation
One pathway (Figure 1) involves the activation of adenylate and protein phosphorylation are important aspects of the human
cyclase and the formation of cAMP, probably coupled with the acrosome reaction. Preliminary data from our laboratory indicate
inhibition of cAMP-phosphodiesterase (which hydrolyses cAMP that at least 14 proteins become phosphorylated in human
to 5'-AMP) (Table I). cAMP activates protein kinase A which spermatozoa, four of which are enriched in the material that is
causes protein phosphorylation (Table II). The other pathway released during the acrosome reaction. Specific phosphoproteins
(Figure 2; phosphoinositide pathway) involves the formation of and their function remain to be identified. In the mouse,
diacylglycerol (DAG) and inositol triphosphate (InsP3) by the phosphorylation of a 45 kDa and a 95 kDa protein occurs during
activation of a phospholipase C which hydrolyses the acrosome reaction or in response to stimulation by a zona
phosphatidylinositol biphosphate (PIPj) (Table HI). DAG and protein (ZP3) (Leyton and Saling, 1989; Suzuki et al., 1990).
Ca 2+ activate protein kinase C (in the presence of phospholipid) The 95 kDa protein may be autophosphorylated tyrosine kinase.
which, similarly to protein kinase A, phosphorylates proteins. In the ram, protein phosphorylation may not take place during
InsP3 can cause the release of Ca 2+ from internal cytoplasmic the acrosome reaction (Roldan and Harrison, 1988).
stores but there is no evidence to suggest that the sperm head Phosphorylation of ATPases is known to inhibit their activity,
has such stores. Support for a role of kinase C in the human which is conducive to the acrosome reaction (see above).
acrosome reaction has also been obtained by others (e.g. Fahmy Phosphorylation may also activate structural proteins or enzymes

Receptor

^ ^ 5-AMP-»
Phosphodiesterase

Protein Kinase A

Phosphorylation
i
i

ACROSOME REACTION

Fig. 1. Proposed cAMP second messenger pathway in the human sperm acrosome reaaion

1270
 Human sperm capacltatton and the acrosome reaction

playing a more direct role in the cytoskeletal changes which are the cytoskeleton which hold the vesicle in place, so that the vesicle
part of the acrosome reaction. In somatic cells, evidence is membrane can come in contact with the plasma membrane at
accumulating that proteins which cause the fusion of the vesicle the sites where the fusion proteins are located. After contact, the
with the plasma membrane must be activated. In one model, these fusion proteins of the vesicle membrane become embedded in
fusion proteins are located at particular sites on the vesicle the plasma membrane and open to form channels. Phospholipids
membrane (Burgoyne, 1990). When the fusion proteins are from the vesicle and plasma membranes enter each channel and
activated, they overcome the action of the structural proteins of mix, resulting in the fusion of the two membranes at that site.
A possible model for such fusion was presented by
Hammerstedt et al. (1990). Membrane phospholipids have a
preferential orientation (head groups outward, hydrocarbon tails
Table I. Effect of adenylate cyclase (AC) stimulator and inhibitors on the
human sperm acrosome reaction inward). Rearrangement of this orientation (head groups inward,
tails outward, termed hexagonal phase H) results in a weak
AC inhibitor AC stimulator % Acrosome reaction
permeability barrier for polar molecules. Thus, the occurrence
None None 8 ± 5 of hexagonal phase II allows the reorientation of lipid and protein
None Forskolin (10 jiM) 39 ± 6 molecules in the two membranes, resulting in fusion. Cleavage
Adenosine (1 mM) Forskolin (10/iM) 17 ± 6
at the fusion site or the formation of fusion pores results in a
2'-0-Methyladenosine (1 mM) Forskolin (10 fiM) 12 ± 4
2',3'-DKleoxyadenosine (1 mM) Forskolin (10 /iM) 14 ± 3 connection between the intra-vesicular space and the extracellular
space (Aimers, 1990). In the case of the acrosome, a number
After a 3 h capacitation period, the spermatozoa were either treated with of such pores along the surface of the OAM and surrounding
stimulator for 15 min before stopping the reaction and determining the
percentage of acrosome-reacted spermatozoa, or were incubated for 5 min
plasma membrane would result in the vesiculation and
with inhibitor before 15 mm treatment with activator. The percentage of disappearance of the membranes. However, it remains to be
motile spermatozoa did not change significantly during treatment, established whether any of these events are actually associated
(n = 3-4). For further details, see De Jonge el al. 1991b.
with the acrosome reaction.

Other enzymes involved in the acrosome reaction

Table II. Effect of protein kinase A (PKA) activators and inhibitors on the Spermatozoa (including those of the human) contain phospho-
human sperm acrosome reaction
lipase A2 which hydrolyses membrane phospholipids to free
PKA inhibitor PKA stimulator % Acrosome reaction fatty acids (e.g. arachidonic acid) and lysophospholipids (Figure
None None- 9 ± 4 3). Localized digestion of membrane lipids by phospholipases
None dbcAMP (1 mM) 37 ± 6 may alter the lipid composition and weaken the membrane,
None 8-bromo cAMP (1 mM) 40 ± 4 producing fusogenic areas. Furthermore, arachidonic acid and
Walsh (10 ^M) dbcAMP (1 mM) 11 ± 1 lysophospholipids induce the acrosome reaction when added to
H-8 (10 fiM) dbcAMP (1 mM) 13 ± 5 capacitated human and non-human spermatozoa. Both lipids may
See Table 1 for brief experimental protocol. The percentage of motile have a direct effect on the structural integrity of the lipid bilayer.
spermatozoa did not change significantly during treatment (n = 3). Arachidonic acid can also act via its metabolites (see below) or
dbcAMP = dibutyryl cyclic AMP, Walsh = Walsh PKA inhibitor from stimulate protein kinase C. Phospholipase A2 can be activated
rabbit muscle; H-8 = A'-[2-(methylamino) ethyl]-5-isoquinolinesulphonamide
dihydrochloride. For further details, see De Jonge el al., 1991b. via receptor mediated-stimulation of G-proteins. Phospholipase

Diacylglycerol Inositol triphosphate

Lipase Ca'

Arachidonic acid Protein Kinase C

\
I
Phosphorylation
\ i

ACROSOME REACTION
Fig. 2. Proposed phosphoinositide second messenger pathway in the human sperm acrosome reaction.

1271
L J.D.Zaneveld et al.

A2 inhibitors prevent the acrosome reaction of rodent sper- exogenous prostaglandins can stimulate the acrosome reaction
matozoa indicating that the enzyme has an important role in these of human spermatozoa.
species. However, such inhibitors have little or no effect on the Acrosin has an essential role in the acrosome reaction, judging
human sperm acrosome reaction at concentrations that should from the inhibitory effect of proteinase inhibitors. Acrosin may
inhibit the enzyme, so that it does not appear to be a rate-limiting act by several mechanisms. In non-human species, proteinase
enzyme in the human (Anderson et al., 1990b). inhibitors prevent the dispersion of the acrosomal matrix,
Arachidonic acid can also be formed by hydrolysis of indicating that one of the actions of acrosin is the lysis of the
diacylglycerol (DAG) through the action of a lipase (Figure 3). structural components of the acrosome. Whether acrosin also has
Arachidonic acid is metabolized by cyclooxygenase, ultimately a role in membrane fusion is arguable. Serine proteinases can
producing prostaglandins, prostacyclins and thromboxanes and activate phospholipases A2 and C, and adenylate cyclase so that
by lipoxygenase, producing hydroxyeicosatetraenoic acids another function of acrosin may be the activation of such
(HPETEs and HETEs) and leukotrienes (Figure 3). In rodents regulatory enzymes. However, since inhibitors of acrosin can
and some other species, several of these metabolites induce the prevent the dbcAMP-induced acrosome reaction of human
acrosome reaction when added to capacitated spermatozoa, spermatozoa (De Jonge et al., 1989), it is likely that acrosin exerts
whereas cytclooxygenase and lipoxygenase inhibitors prevent the its effect after adenylate cyclase activation. Ram proacrosin can
reaction. This suggests an essential role of endogenous bind to and be activated by calmodulin and calmodulin is rapidly
arachidonic acid metabolites in the acrosome reaction of some hydrolysed by acrosin (Frenette etai, 1990). Proteinase
non-human spermatozoa. Their mechanism of action is not known inhibitors can prevent the progesterone-stimulated rise in
although some metabolites may stimulate Ca 2+ transport. Recent intracellular Ca 2+ of human spermatozoa (Pillai and Meizel,
data from our laboratory indicate that the human sperm acrosome 1990). Thus, acrosin may also be in some way involved in the
reaction is not prevented by cyclooxygenase or lipoxygenase Ca 2+ transport mechanism.
inhibitors; thus endogenous arachidonic acid metabolites do not
appear to have an essential role (if any) in this species. However, Metabolism
The presence or absence of certain sugars and other metabolic
Tabte m . Effect of protein kinase C (PKC) activators and inhibitor on the substrates in the medium can influence human sperm capacitation
human sperm acrosome reaction and the acrosome reaction (e.g. Rogers and Perreault, 1990).
PKC inhibitor PKC activator % Acrosome reaction
This may be in part be due to an inhibitory or activating effect
of these agents on the enzyme systems discussed above. Another
None None 1 ± 2 explanation is that certain metabolic processes, e.g. resulting in
None 4/3-PDD (0.1 /*M) 34 ± 4
None PMA (0.1 nM) 30 ± 5 the generation of ATP, are required for the maintainance of
None OAG (50 ^M) 27 ± 3 acrosomal integrity and/or for the induction of the acrosome
None DOG (50 fiM) 31 ± 3 reaction. This is not surprising since ATP is required for the
H-7 (1 jiM) DOG (50 /iM) 11 ± 1 activity of the ATPases, for cAMP formation, for phosphory-
See Table I for brief experimental protocol. The percentage of motile
lation, etc.
spermatozoa did not change significantly during treatment (n = 3). Metabolic processes occur in the sperm tail and are known
4(3-PDD = active isomer of phorbol 12,13-didecanoate; PMA = phorbol to change during capacitation and/or the acrosome reaction. These
12-myristate 13-acetatc; OAG = l-oleoyl-2-acetyl-sn-glycerol,
DOG =• 1,2-dioctanoyl-sn-glycerol; H-7 = l-(5-isoquinolinylsulphonyl- metabolic changes are ultimately expressed in a rapid increase
2-methylpiperazine). For further details see De Jonge el al., 1991a and alteration in tail movements, called 'hyperactivated' motility.

Membrane
Phospholipids

Diacylglycerol
Phospholipase A 2
Lipase

Arachidonic acid Lysophospholipids

Cyclooxygenase Lipoxygenase

Prostaglandins H-eicosatetraenoic acids

Prostacyclins Leukotrienes
Thromboxanes

Fig. 3. Action of phospholipase A2, and the formation and metabolism of arachidonic acid.
1272
 Human sperm capadtation and the acrosome reaction

It has been suggested that the metabolic activity of the tail Much of the foregoing review is speculative and mostly based
somehow effects the acrosome reaction process. However, there on conjecture rather than on well-defined research. However,
is no evidence that such communication exists between the tail the model should aid in the design of experiments which can
and the head and hyperactivated motility can occur independently prove or disprove the proposed mechanisms.
of the acrosome reaction. Furthermore, recent results from our
laboratory suggest that the acrosome reaction can be induced by Acknowledgements
calcium ionophore in tailless spermatozoa. Thus, it is more likely The authors appreciate the manuscript preparation by Ms C.Griffin.
Unpublished results described in this review were from work supported
that the sperm head itself can metabolize certain sugars and that
by N1H HD 19555.
the metabolic products can influence the acrosome reaction.
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Received on April 4, 1991; accepted on July 18, 1991