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
Departments of Obstetrics and Gynecology, Biochemistry and
Physiology, Rush University, Rush-Presbyterian-St. Luke's Medical
Center, Chicago, IL 60612, USA
'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 mem-
brane 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
Key words: spermatozoa/acrosome/human/G-proteins/protein
phosphorylation
Introduction
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 uncertain-
ty as to which are physiological. The observations often appear
to be unrelated to each other so that it has been difficult to com-
bine 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 sec-
tions of the plasma membrane with the OAM, the lysis/dis-
appearance 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 pro-
acrosin activation occurs during capacitation but as the acrosome
reaction takes place, a large proportion of the proacrosin is con-
verted 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. Acrosome-
reacted 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

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
Human sperm capacitation and the acrosome reaction
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 carbo-
hydrate moiety of the glycocalyx may further explain the altera-
tions 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 struc-
tural 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 phospho-
lipids causes destabilization of the membrane and an increase in
membrane permeability. Thus, it is not surprising that under in-
vitro 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

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 <5.0, at
least in rodent spermatozoa). Antiport systems may also be
present. Different ATPases appear to be associated with the
plasma membrane and 0AM so that the ionic composition of
the cytosolic and intra-acrosomal spaces may vary.
Inhibition of any one of these three ATPases has been shown
to cause the acrosome reaction of rodent spermatozoa. Whether
or not this is true for human spermatozoa remains to be
Human sperm capacitation and the acrosome reaction
established. Physiologically, it is possible that these pumps are
either overwhelmed or inhibited during the early stages of the
acrosome reaction, leading to an influx of Ca 2+ and Na + and
a decrease in intra-acrosomal H + . Changes in these and other
ions may alter the osmotic pressure of the sperm head
compartments and cause a rise in acrosomal pH. The rise in pH
may cause the activation of enzymes, for instance that of
proacrosin to acrosin. Inhibition of the ATPases would also
decrease ATP utilization. ATP is the substrate for adenylate
cyclase, an enzyme that appears to be regulatory in the acrosome
reaction (see below).
The mechansim whereby high cytosolic Ca 2+ can induce the
acrosome reaction or exocytosis in somatic cells is poorly
understood. In the simplest fusion model (phospholipid vesicles
and a planar phospholipid membrane), Ca 2+ can overcome the
repulsive forces (electrostatic and hydration barriers) between
the two membranes by interacting with the polar phospholipid
head groups (Niles and Cohen, 1991). This allows adhesion to
occur but does not lead to fusion unless the osmotic pressure in
the vesicle is increased, causing swelling of the vesicle. Since
the acrosome reaction occurs with an increase in cytosolic Ca 2+ ,
possibly coupled with an increase in intra-acrosomal osmotic
pressure and swelling of the acrosome, it is conceivable that this
simple mechanism is sufficient to explain the acrosome reaction.
However, it is generally thought that, at least in somatic cells,
the effects of Ca 2+ are less direct. Ca 2+ may act as a second
messenger and activate certain enzyme systems. For instance,
phosphoinositide breakdown and protein kinase C activity (see
below) require Ca 2+ (e.g. Roldan and Harrison, 1989). Ca 2+
may also complex with calmodulin which, in turn, can activate
Ca2+-calmodulin protein kinase which stimulates protein
phosphorylation. Calmodulin binding to and activation of
proacrosin have been reported (Frenette et al., 1990). Finally,
Ca 2+ may directly activate proteins that cause the fusion of the
0AM with the plasma membrane and/or aid in overcoming the
cytoskeletal forces that hold the 0AM and plasma membrane in
place.
Second messenger systems and protein phosphorylation
The mechanism of exocytosis may vary between different somatic
cell types and several mechanisms may be employed by the same
cell. Both Ca2+-dependent and independent mechanisms have
been found. In certain cells, Ca 2+ merely acts as a cofactor
which accelerates the exocytotic response. A common mechanism
in these cases is the membrane receptor activation of GTP-binding
proteins (G-proteins) which in turn stimulate second messenger
systems and regulate ion transport mechanisms. Mechanisms can
change with the availability of stimulatory components. For
instance, normally both diacylglycerol and Ca 2+ are required
for the activation of protein kinase C (in the presence of
phospholipid) but high intracellular Ca 2+ levels as a result of
calcium ionophore treatment of the cell are alone sufficient for
the activation of the enzyme.
In somatic cells, a large number of signals are known to act
via G-protein-coupled receptors including neurotransmittors,
peptide and glycoprotein hormones and arachidonic acid
metabolites (prostaglandins, thromboxanes, leukotrienes)
(Birnbaumer, 1990). Data in non-human species are beginning
1269
L.J.D.Zaneveld el at.
to accumulate which suggest that the acrosome reaction is
mediated by G-proteins (Kopf, 1988). Certain G-proteins are
specifically associated with the acrosomal region and several are
located only at the acrosomal tip (Garty et al., 1988). G-proteins
may also have a role in the human sperm acrosome reaction.
Cholera toxin (activates Gs which stimulates the adenylate
cyclase system) and pertussis toxin (inactivates several G-proteins,
including the G, which exerts an inhibitory effect on the
adenylate cyclase system) both stimulate the human acrosome
reaction (Anderson et al., 1991). Ca 2+ was not required for the
effect of the toxins. However, these data are still controversial
because Lee et al. (1990) reported that the zona-induced
acrosome reaction of human spermatozoa is inhibited by pertussis
toxin. Furthermore, coupling of G-proteins with the adenylate
cyclase system of spermatozoa is not definite (e.g. Hildebrandt
etai, 1985).
One of the functions of G-proteins is the activation of second
messenger systems. Until recently, little information was available
on the role of second messenger systems in the acrosome reaction.
However, it now appears that at least two such pathways func-
tion in this reaction in the human (De Jonge et al., 1991a,b).
One pathway (Figure 1) involves the activation of adenylate
cyclase and the formation of cAMP, probably coupled with the
inhibition of cAMP-phosphodiesterase (which hydrolyses cAMP
to 5'-AMP) (Table I). cAMP activates protein kinase A which
causes protein phosphorylation (Table II). The other pathway
(Figure 2; phosphoinositide pathway) involves the formation of
diacylglycerol (DAG) and inositol triphosphate (InsP3) by the
activation of a phospholipase C which hydrolyses
phosphatidylinositol biphosphate (PIPj) (Table HI). DAG and
Ca 2+ activate protein kinase C (in the presence of phospholipid)
which, similarly to protein kinase A, phosphorylates proteins.
InsP3 can cause the release of Ca 2+ from internal cytoplasmic
stores but there is no evidence to suggest that the sperm head
has such stores. Support for a role of kinase C in the human
acrosome reaction has also been obtained by others (e.g. Fahmy
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
et al., 1990). Furthermore, kinase C activity has been found in
human spermatozoa (Rotem et al., 1990; unpublished data from
our laboratory) and PIP2 hydrolysis takes place when human
spermatozoa are treated with acrosome reaction stimulators
(Thomas and Meizel, 1989). However, species differences may
exist; for instance, no protein kinase C was detected in ram
spermatozoa (Roldan and Harrison, 1988).
Other second messenger systems and protein kinases may also
be involved in the acrosome reaction, such as cGMP-dependent
kinase and Ca2+-calmodulin kinase but this is mostly speculative
at the present time. More evidence is available for a role of
tyrosine kinase in the acrosome reaction (Saling, 1991). In
contrast to the previous kinases, tyrosine kinase may be associated
with the cell surface and can act as a receptor for external ligands
(signals). Recent data suggest that the zona-induced acrosome
reaction of mouse spermatozoa is mediated by tyrosine kinase
(Leyton and Saling, 1989). In somatic cells, tyrosine kinase can
also activate phospholipase C so that the phosphoinositide system
can be stimulated without requiring the action of G-proteins
(Figure 2).
Based on these observations, it appears that kinase activation
and protein phosphorylation are important aspects of the human
acrosome reaction. Preliminary data from our laboratory indicate
that at least 14 proteins become phosphorylated in human
spermatozoa, four of which are enriched in the material that is
released during the acrosome reaction. Specific phosphoproteins
and their function remain to be identified. In the mouse,
phosphorylation of a 45 kDa and a 95 kDa protein occurs during
the acrosome reaction or in response to stimulation by a zona
protein (ZP3) (Leyton and Saling, 1989; Suzuki et al., 1990).
The 95 kDa protein may be autophosphorylated tyrosine kinase.
In the ram, protein phosphorylation may not take place during
the acrosome reaction (Roldan and Harrison, 1988).
Phosphorylation of ATPases is known to inhibit their activity,
which is conducive to the acrosome reaction (see above).
Phosphorylation may also activate structural proteins or enzymes
 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-
matozoa indicating that the enzyme has an important role in these
species. However, such inhibitors have little or no effect on the
human sperm acrosome reaction at concentrations that should
inhibit the enzyme, so that it does not appear to be a rate-limiting
enzyme in the human (Anderson et al., 1990b).
Arachidonic acid can also be formed by hydrolysis of
diacylglycerol (DAG) through the action of a lipase (Figure 3).
Arachidonic acid is metabolized by cyclooxygenase, ultimately
producing prostaglandins, prostacyclins and thromboxanes and
by lipoxygenase, producing hydroxyeicosatetraenoic acids
(HPETEs and HETEs) and leukotrienes (Figure 3). In rodents
and some other species, several of these metabolites induce the
acrosome reaction when added to capacitated spermatozoa,
whereas cytclooxygenase and lipoxygenase inhibitors prevent the
reaction. This suggests an essential role of endogenous
arachidonic acid metabolites in the acrosome reaction of some
non-human spermatozoa. Their mechanism of action is not known
although some metabolites may stimulate Ca 2+ transport. Recent
data from our laboratory indicate that the human sperm acrosome
reaction is not prevented by cyclooxygenase or lipoxygenase
inhibitors; thus endogenous arachidonic acid metabolites do not
appear to have an essential role (if any) in this species. However,
Tabte m . Effect of protein kinase C (PKC) activators and inhibitor on the
human sperm acrosome reaction
PKC inhibitor PKC activator % Acrosome reaction
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
None OAG (50 ^M) 27 ± 3
None DOG (50 fiM) 31 ± 3
H-7 (1 jiM) DOG (50 /iM) 11 ± 1
See Table I for brief experimental protocol. The percentage of motile
spermatozoa did not change significantly during treatment (n = 3).
4(3-PDD = active isomer of phorbol 12,13-didecanoate; PMA = phorbol
12-myristate 13-acetatc; OAG = l-oleoyl-2-acetyl-sn-glycerol,
DOG =• 1,2-dioctanoyl-sn-glycerol; H-7 = l-(5-isoquinolinylsulphonyl-
2-methylpiperazine). For further details see De Jonge el al., 1991a
exogenous prostaglandins can stimulate the acrosome reaction
of human spermatozoa.
Acrosin has an essential role in the acrosome reaction, judging
from the inhibitory effect of proteinase inhibitors. Acrosin may
act by several mechanisms. In non-human species, proteinase
inhibitors prevent the dispersion of the acrosomal matrix,
indicating that one of the actions of acrosin is the lysis of the
structural components of the acrosome. Whether acrosin also has
a role in membrane fusion is arguable. Serine proteinases can
activate phospholipases A2 and C, and adenylate cyclase so that
another function of acrosin may be the activation of such
regulatory enzymes. However, since inhibitors of acrosin can
prevent the dbcAMP-induced acrosome reaction of human
spermatozoa (De Jonge et al., 1989), it is likely that acrosin exerts
its effect after adenylate cyclase activation. Ram proacrosin can
bind to and be activated by calmodulin and calmodulin is rapidly
hydrolysed by acrosin (Frenette etai, 1990). Proteinase
inhibitors can prevent the progesterone-stimulated rise in
intracellular Ca 2+ of human spermatozoa (Pillai and Meizel,
1990). Thus, acrosin may also be in some way involved in the
Ca 2+ transport mechanism.
Metabolism
The presence or absence of certain sugars and other metabolic
substrates in the medium can influence human sperm capacitation
and the acrosome reaction (e.g. Rogers and Perreault, 1990).
This may be in part be due to an inhibitory or activating effect
of these agents on the enzyme systems discussed above. Another
the generation of ATP, are required for the maintainance of
acrosomal integrity and/or for the induction of the acrosome
reaction. This is not surprising since ATP is required for the
activity of the ATPases, for cAMP formation, for phosphory-
lation, etc.
Metabolic processes occur in the sperm tail and are known
to change during capacitation and/or the acrosome reaction. These
metabolic changes are ultimately expressed in a rapid increase
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

It has been suggested that the metabolic activity of the tail
somehow effects the acrosome reaction process. However, there
is no evidence that such communication exists between the tail
and the head and hyperactivated motility can occur independently
of the acrosome reaction. Furthermore, recent results from our
laboratory suggest that the acrosome reaction can be induced by
calcium ionophore in tailless spermatozoa. Thus, it is more likely
that the sperm head itself can metabolize certain sugars and that
the metabolic products can influence the acrosome reaction.
Conclusions
The model for capacitation and the acrosome reaction presented
here proposes that the genital tract possesses a mechanism
whereby the activation and release of acrosin (and/or other
enzymes) from the sperm acrosome is prevented until the
spermatozoon reaches the target site for the action of the enzymes
(the zona pellucida in the case of acrosin). Such regulation is
acomplished by the presence of peripheral factors on the sperm
surface which prevent the occurrence of the acrosome reaction
by blocking receptor sites, ion channels and/or certain enzymes,
or by increasing membrane stability. As the spermatozoa are
transported through the female genital tract (and the cumulus cell
layer), these inhibitory factors are removed (a step in the
capacitation process). The acrosome reaction is initiated as the
spermatozoon approaches the zona pellucida but is not completed
until after binding to the zona has taken place. During the
initiation of the acrosome reaction, proacrosin (the inactive form
of acrosin) is activated. Acrosin is released as the acrosome
reaction occurs, causing the localized digestion of the zona
pellucida.
Several signals may be produced by the oocyte and may cause
the initiation of the acrosome reaction process by activating
receptors on the sperm plasma membrane. Receptor stimulation
may lead to the activation of G-proteins which change the ion
transport activity of the plasma membrane and activate several
second messenger systems. The former results in a change in
the intracellular composition of ions, including an increase in
Ca2 + , and the latter results in the activation of several kinases
and the phosphorylation of proteins. Alternatively, receptor
activation may cause protein phosphorylation directly. These
activated proteins/enzymes and ion changes drive the acrosome
reaction further, ultimately overcoming the cytoskeletal and
osmotic forces that keep the acrosome and plasma membrane
in place. Events that are part of the cascade are, at least in some
nonhuman species, the activation of phospholipase A2 and the
metabolism of arachidonic acid and, in all species, the activa-
tion of proacrosin to acrosin. Exactly where these enzymes and
processes fit in the overall mechanism of the acrosome reaction
is as yet unknown. The function of acrosin appears to be the
digestion of the acrosomal matrix. Localized changes in
membrane lipids occur, probably by a decrease in the cholesterol
phospholipid ratio and a migration of phospholipids and proteins,
resulting in fusogenic regions in the plasma membrane. Fusion
of the plasma membrane and the outer acrosomal membrane
occurs at a number of sites, possibly due to the presence of fusion
proteins. Openings or pores are formed at the fusion sites,
resulting in membrane vesiculation, the disperson of the vesicles
and the release of the acrosomal contents.
Human sperm capadtation and the acrosome reaction
Much of the foregoing review is speculative and mostly based
on conjecture rather than on well-defined research. However,
the model should aid in the design of experiments which can
prove or disprove the proposed mechanisms.
Acknowledgements
The authors appreciate the manuscript preparation by Ms C.Griffin.
Unpublished results described in this review were from work supported
by N1H HD 19555.
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Received on April 4, 1991; accepted on July 18, 1991