Journal of Cell Science 104, 163-172 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
163
Proteases are not involved in the membrane fusion events of the
lysolecithin-mediated guinea pig sperm acrosome reaction
Sean P. Flaherty* and Nicholas J. Swann
Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South
Australia 5011, Australia
*Author for correspondence
SUMMARY
The guinea pig sperm acrosome reaction is characterized by a complex temporal and structural pattern of
membrane fusions. In this study, we have used specific
protease inhibitors to determine if proteases regulate
this pattern of membrane fusions during the
lysolecithin-mediated guinea pig sperm acrosome reaction. Inhibitors were chosen so as to cover a wide range
of different types of proteases, and all were used at the
highest concentration that did not adversely affect
sperm motility. Of the eight inhibitors tested, leupeptin,
soya bean trypsin inhibitor (SBTI), p-aminobenzamidine (pAB) and nitrophenyl p -guanidino benzoate
(NPGB) inhibited completion of the acrosome reaction,
while diethylenetriaminepentaacetic acid (DTPA), phosphoramidon, bestatin and pepstatin had no effect.
Sperm that had been acrosome-reacted in the presence
of each inhibitor were examined by transmission electron microscopy to assess whether the inhibitors altered
INTRODUCTION
The mammalian sperm acrosome reaction is an exocytotic
event that occurs prior to or during sperm penetration of
the zona pellucida around the oocyte. It is characterized by
multiple fusions between the plasma membrane and outer
acrosomal membrane over the apical and principal segments of the acrosome (Barros et al., 1967; Russell et al.,
1979). The acrosomal matrix then disperses, releasing
hydrolytic enzymes which facilitate penetration through the
cumulus oophorus and zona pellucida (Talbot, 1985;
Yanagimachi, 1988). We recently described a complex
structural and temporal pattern of membrane fusions which
occurs during the lysolecithin-mediated acrosome reaction
of guinea pig spermatozoa (Flaherty and Olson, 1988,
1991). Specific domains of the plasma membrane and outer
acrosomal membrane were non-fusigenic, while other
regions exhibited unique patterns of fusion. Membraneassociated cytoskeletal elements imparted a directional
component to membrane fusion on the dorsal aspect of the
apical segment. This pattern of fusions was evident in sperm
the pattern of membrane fusions during the acrosome
reaction. DTPA, phosphoramidon, bestatin and pepstatin had no effect on membrane fusion or matrix dispersal. Serine protease inhibitors such as leupeptin,
SBTI, pAB and NPGB prevented complete dispersal of
the acrosomal matrix and completion of the acrosome
reaction, but did not alter the temporal sequence or
structural pattern of membrane fusions. The undispersed matrix was present along the dorsal and ventral
aspects of the apical segment and throughout the principal segment. We conclude that proteases are not
involved in regulating the temporal and structural pattern of membrane fusions which occurs during the
lysolecithin-mediated acrosome reaction of guinea pig
sperm.
Key words: sperm, acrosome reaction, membranes, fusion,
proteases
that acrosome-reacted in rouleaux (Flaherty and Olson,
1988) and in single sperm that had been dissociated from
rouleaux prior to induction of the acrosome reaction (Flaherty and Olson, 1991).
It has been suggested that proteases are involved in membrane fusion processes (Lucy, 1984; Lennarz and Strittmatter, 1991). Studies on myoblast fusion and exocytosis in
mast cells and adrenal chromaffin cells have suggested that
proteolysis of membrane proteins by metalloendoproteases
is required for membrane fusion (Couch and Strittmatter,
1983; Mundy and Strittmatter, 1985) and Farach et al.
(1987) proposed that metalloendoproteases are involved in
the sea urchin sperm acrosome reaction. There is also evidence for the involvement of proteolytic activity in the
mammalian sperm acrosome reaction. Meizel and co-workers (Dravland et al., 1984; Meizel, 1984) presented evidence
that trypsin-like proteases are involved in the membrane
fusion events of the hamster sperm acrosome reaction.
However, other studies on mouse and guinea pig sperm
have shown that trypsin-like protease activity is only
required for dispersal of the acrosomal matrix and is not
164
S. P. Flaherty and N. J. Swann
involved in the membrane fusion events (Green, 1978a;
Fraser, 1982; Perreault et al., 1982; Huang et al., 1985).
On the basis of this evidence, as well as studies showing that acrosomal components are sequestered in specific
domains of the acrosomal matrix (Huang et al., 1985;
Talbot and DiCarlantonio, 1985; Olson et al., 1988; Noland
et al., 1989; Hardy et al., 1991), we put forward the hypothesis that proteases might exert a localized effect on specific
domains of the plasma membrane and outer acrosomal
membrane, thus regulating the structural pattern of membrane fusions during the guinea pig sperm acrosome reaction. We have tested this hypothesis using specific protease
inhibitors. However, in contrast to previous studies, which
concentrated on the role of serine proteases in the guinea
pig sperm acrosome reaction (Green, 1978a; Perreault et
al., 1982; Huang et al., 1985), we used a range of different inhibitors so as to encompass the different proteases that
may be localized in the sperm acrosome (Harrison, 1983).
Sperm were induced to undergo the acrosome reaction
using lysolecithin (Fleming and Yanagimachi, 1981; Flaherty and Olson, 1988), so that a high percentage of synchronous acrosome reactions would be obtained, and sperm
were examined by transmission electron microscopy to
determine whether the inhibitors changed the temporal or
structural pattern of fusions during the acrosome reaction.
We found that serine protease inhibitors prevented dispersion of the acrosomal matrix as reported previously (Green,
1978a; Fraser, 1982; Perreault et al., 1982; Huang et al.,
1985), but none of the inhibitors altered the structural pattern of fusions.
MATERIALS AND METHODS
Chemicals
Chemicals and their suppliers were as follows: leupeptin, pepstatin, phosphoramidon, bestatin (Boehringer-Mannheim,
Mannheim, Germany); soya bean trypsin inhibitor (SBTI, Type IS), p-aminobenzamidine (pAB), diethylenetriaminepentaacetic
acid (DTPA), nitrophenyl p′-guanidino benzoate (NPGB), bovine
serum albumin (BSA, A-7030), fatty acid-free BSA (A-6003),
lysolecithin (lysophosphatidyl choline, palmitoyl; LPC) (Sigma
Chemical Co, St Louis, MO, USA); tannic acid (Mallinckrodt,
Paris, KY, USA); other electron microscopy reagents (BioRad,
Richmond, CA, USA; Probing and Structure, Thuringowa Central, Queensland, Australia); NembutalR (sodium pentobarbitone)
(Boehringer-Ingelheim, Artarmon, NSW, Australia).
Culture media
A modified Tyrode’s solution was used (Fleming and Yanagimachi, 1981; Flaherty and Olson, 1991). Ca2+-deficient, Hepesbuffered medium (HmT) was used for sperm preparation, Ca2+deficient medium (mT) was used for sperm incubations and 2 ×
Ca2+ mT (CmT) was added to initiate the acrosome reaction.
Protease inhibitors
Preliminary experiments were performed to determine the maximum concentration of each inhibitor that could be used without
adversely affecting sperm motility; 70% motility was considered
acceptable. These inhibitor concentrations were used in all subsequent experiments (see Table 1). DTPA, phosphoramidon and
leupeptin were prepared as stock solutions in distilled water and
stored at −65˚C. SBTI and pAB were prepared fresh in HmT and
Table 1. The specificity and concentration of the
protease inhibitors used
Inhibitor
DTPA
Phosphoramidon
Leupeptin
Pepstatin
Bestatin
SBTI
pAB
NPGB
Final
concentration
100 µg/ml
500 µg/ml
200 µg/ml
100 µg/ml
500 µg/ml
5 mg/ml
1 mg/ml
50 µg/ml
Specificity
Metalloendoproteases
Metalloendoproteases
Serine and thiol proteases
Acid proteases
Exopeptidases
Trypsin and acrosin
Serine proteases
Trypsin
the pH was adjusted to 7.5. Bestatin and pepstatin were stored at
−65˚C as stock solutions in methanol, while NPGB was prepared
fresh in DMSO. The concentrations of methanol (<5%) and
DMSO (<1%) in the media did not affect sperm viability or the
acrosome reaction.
Sperm preparation and capacitation
Adult male Dunkin-Hartley guinea pigs (>700 g; Therapeutic
Goods Administration Laboratories, Woden, ACT, Australia) were
killed by an overdose of NembutalR and sperm were flushed from
the distal cauda epididymis and vas by retrograde infusion of
warm HmT. The sperm concentration was adjusted to 100 × 106
sperm/ml in HmT. Sperm were then diluted to 10 × 106 sperm/ml
in mT containing 80 µg/ml LPC and incubated at 37°C in a dry
block heater for 60-70 min to effect capacitation.
Effect of protease inhibitors on the acrosome
reaction
The first series of experiments were performed to determine the
effects of various inhibitors on the occurrence of the acrosome
reaction. After 60-70 min in lysolecithin, an appropriate amount
of inhibitor was added and the tubes were incubated at 37°C for
10 min. An equal volume of CmT was then added to each tube
and they were incubated for 15 min at 37°C. Immediately before
addition of Ca2+, and at 5 and 15 min post-Ca2+, a 25 µl sample
was removed and added to 100 µl of 3% glutaraldehyde in 0.1 M
cacodylate buffer to stop the acrosome reaction. Samples were
coded and scored blind for occurrence of the acrosome reaction
using phase-contrast microscopy. Five replicates were performed
and the mean values are presented.
Ultrastructural studies on the effect of inhibitors on the membrane fusion events of the acrosome reaction were carried out
using the above protocol. Three replicates were performed. At 45
s and 1, 1.25, 2, 3.5, 5 and 10 min after the addition of Ca2+,
sperm were fixed by the addition of an equal volume of cold 5%
glutaraldehyde in 0.15 M cacodylate buffer. They were then pelleted and processed for transmission electron microscopy as
described previously (Flaherty and Olson, 1988, 1991). Ultrathin
sections were contrasted with uranyl acetate and lead citrate and
examined using a Jeol 100s electron microscope.
RESULTS
Effect of protease inhibitors on occurrence of the
acrosome reaction
The effect of protease inhibitors on acrosomal status and
induction of the acrosome reaction is shown in Fig. 1.
Sperm motility was maintained at 70-80% throughout the
incubations. Fig. 1A illustrates that >80% of the sperm were
Role of proteases in sperm acrosome reaction
165
A
100
75
50
25
0
H2 O
B
100
75
50
25
0
H 2O
C
100
75
50
25
0
H2O
Fig. 1. The percentages of acrosome-intact,
partially acrosome-reacted and completely
acrosome-reacted sperm in the inhibitor and
control groups: (A) after 10 min in
inhibitors, (B) 5 min after adding calcium
and (C) 15 min after adding calcium. Mean
values of 5 experiments are shown.
Standard errors were always less than 7%
of sperm (usually 1-4%) for any sperm
category in a given treatment group.
166
S. P. Flaherty and N. J. Swann
Figs 2-4. Stages 1 to 3 of the guinea pig sperm acrosome reaction.
Fig. 2. Stage 1. Fusion between the plasma membrane and outer acrosomal membrane has commenced on the antero-ventral and dorsal
surfaces of the apical segment (arrows). The acrosomal matrix has cavitated in the apical segment (asterisk). Bar, 1 µm.
Fig. 3. Fusion on the dorsal (convex) surface of the apical segment gives rise to hybrid membrane sheets (s) and parallel hybrid
membrane tubules (t). The orientation of filaments on the luminal surface of the outer acrosomal membrane in the sheets and tubules is
indicated by the arrows. Bar, 0.5 µm.
Fig. 4. Stages 2 and 3. (A) One spermatozoon at stage 2 (2) and another at stage 3 (3). In stage 2, fusion is almost complete in the apical
segment (as), and is spreading to the principal segment (ps), which is already undergoing cavitation. There is a thick layer of undispersed
matrix on the ventral (concave) surface of the apical segment (**) and another layer on the dorsal surface (*). In stage 3, the tubular
pattern of fusion has spread throughout the principal segment (ps) and very little matrix remains in the apical segment (as). es, equatorial
segment; s, hybrid membrane sheets. (B) A higher magnification of cavitation and initial fusion in the principal segment (ps) during stage
2. es, equatorial segment. (C) Illustrates the random tubular pattern of fusion in the principal segment during stage 3. Bars, 0.5 µm.
Role of proteases in sperm acrosome reaction
acrosome-intact in each treatment group after capacitation
in lysolecithin and incubation in protease inhibitors. When
scored 5 min post-Ca2+, in >90% of sperm in each treatment group (except pAB) the acrosome reaction had commenced (Fig. 1B). It should be noted that some of the partially acrosome-reacted sperm (about 20%) were degenerate
cells with swollen or damaged acrosomes, so the true level
of induction of the acrosome reaction was about 70%. In
the DTPA, phosphoramidon, bestatin and pepstatin groups,
60-70% of the sperm had completed the acrosome reaction
after 5 min, whereas in the leupeptin, SBTI and NPGB
groups, the acrosome reaction was incomplete in 90% of
the sperm. pAB was the only inhibitor to prevent the acrosome reaction (in 40% of the sperm); the remainder were
at an incomplete stage of the reaction. A similar distribution of acrosome-intact, acrosome-reacted and partially
acrosome-reacted sperm was found at 15 min post-Ca2+
(Fig. 1C), indicating that leupeptin, SBTI, pAB and NPGB
caused a non-reversible inhibition of completion of the
acrosome reaction over this time period.
Membrane fusion events in the presence of
protease inhibitors
In order to simplify the description of the morphological
events of the acrosome reaction, we have used the results
of our previous studies (Flaherty and Olson, 1988, 1991)
to classify the guinea pig sperm acrosome reaction into the
following 5 stages. (1) Membrane fusion has commenced
on the antero-ventral and dorsal surfaces of the apical segment. Cavitation of the matrix is underway in the apical
segment (Fig. 2). (2) Fusion has occurred on the dorsal
(sheets, parallel tubules and random tubules) and ventral
(random tubules) surfaces of the apical segment and is starting to spread to the principal segment (random tubules).
Cavitation of the matrix is underway in the principal segment and matrix persists in the dorsal and ventral regions
of the apical segment (Figs 3, 4A,B). (3) Vesiculation is
complete in the apical segment except for the non-fusigenic
sheets and parallel tubules; a small amount of matrix
remains. The random tubular pattern of fusion has spread
throughout the principal segment (Fig. 4A,C). (4) Vesiculation of the hybrid membrane tubules in the principal segment is underway (Fig. 5A,B). (5) The hybrid membrane
shroud has been shed and only the unreacted equatorial segment of the acrosome remains. The electron-dense matrix
in the equatorial segment persists (Fig. 6).
The stage of the acrosome reaction at different time
points for each inhibitor and control group is summarized
in Table 2. Two main responses were found: (1) DTPA,
phosphoramidon, bestatin and pepstatin had no effect on
either the temporal or structural pattern of membrane
fusions. There were slight differences in the timing of the
acrosome reaction at various time points in the control and
inhibitor groups, but the acrosome reaction was complete
(stage 5) in each case by 3.5 min after Ca2+ addition. (2)
An incomplete acrosome reaction was observed in sperm
incubated with leupeptin, SBTI, pAB and NPGB. The temporal sequence and structural pattern of fusions were not
different from controls, but the acrosome reaction arrested
at stage 3 or 4 and did not progress beyond stage 4 even
at the 10 min time point. This was associated with incom-
167
plete dispersal of the acrosomal matrix in the apical and
principal segments (Fig. 7). Tannic acid fixation accentuated the undispersed matrix components.
Distinct layers of acrosomal matrix persisted in the dorsal
and ventral regions of the apical segment. The undispersed
layer in the ventral region was closely apposed to the outer
acrosomal membrane or the fused hybrid membrane complex (Fig. 7A, C), and although we did not observe any
periodic connections between the matrix and membrane, the
outer acrosomal membrane did exhibit localized regions of
increased electron density, which were absent in other
regions of the acrosome (Fig. 7C). In contrast, the dorsal
matrix layer was usually disconnected from the hybrid
membranes; it was less electron dense and was associated
with smaller matrix foci of similar electron density to the
ventral matrix layer (Fig. 7A). In the principal segment, the
hybrid membrane tubules/vesicles were closely attached to
the inner acrosomal membrane by the undispersed matrix
(Fig. 7B).
DISCUSSION
The role of proteases in the membrane fusion
events of the acrosome reaction
We previously reported that a defined temporal and structural pattern of membrane fusions occurs during the guinea
pig sperm acrosome reaction (Flaherty and Olson, 1988,
1991). A clear understanding of the mechanisms which regulate the pattern of fusions is yet to be obtained. As there
is evidence that proteolysis may be involved in the membrane fusion events of the acrosome reaction (Dravland et
al., 1984; Farach et al., 1987), we tested the hypothesis that
proteases may exert a localized effect on specific domains
of the plasma membrane and outer acrosomal membrane,
thus regulating the structural pattern of membrane fusions
during the guinea pig sperm acrosome reaction. We tested
this hypothesis using inhibitors to a range of proteases, but
found that none of the inhibitors exhibited any effect on the
Table 2. The ultrastructural features of the acrosome
reaction in the presence of protease inhibitors
Stage of AR at indicated time point
Treatment
45 s
1.25 min 2 min 3.5 min
Controls
Water
Methanol
DMSO
1/2
1/2
1/2
2-4
3/4
2/3
4/5
4/5
4
Inhibitors
DTPA
Phosphoramidon
Bestatin
Leupeptin
Pepstatin
SBTI
pAB
NPGB
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/2
2-4
1-3
3/4
2
2/3
2/3
2/3*
1-3*
2-4
3/4
4/5
2/3*
4/5
3/4*
3*
3*
5 min
10 min
5
5
5
5
5
5
5
5
5
5
5
5
3/4*
5
3/4*
3/4*
3/4*
5
5
5
4*
5
3/4*
3/4*
4*
5
5
5
4*
5
3/4*
3/4*
4*
The numbers 1-5 refer to the stages of the acrosome reaction described
in Results. Each data point is derived from three separate experiments.
*Incomplete dispersal of the acrosomal matrix.
168
S. P. Flaherty and N. J. Swann
Figs 5, 6. Stages 4 and 5 of the guinea pig sperm acrosome reaction.
Fig. 5. Stage 4. (A) The hybrid membrane tubules in the principal segment (ps) have vesiculated. Fusion on the ventral surface of the
apical segment (as) has produced vesicles (v), while hybrid membrane sheets (s) and parallel tubules (t) are present on the dorsal surface.
Note that there is little matrix left in the apical segment. (B) Higher magnification of the vesicles (v) in the principal segment. im, inner
acrosomal membrane. Bars, 0.5 µm.
Fig. 6. Stage 5. The hybrid membrane vesicles (v) are shed from the sperm head, exposing the inner acrosomal membrane (im). The
equatorial segment (es) of the acrosome is unreacted and its electron dense matrix persists. pa, postacrosomal region. Bar, 0.25 µm.
structural pattern of fusions during the acrosome reaction.
This confirms previous reports on the effect of serine protease inhibitors (Green, 1978a; Perreault et al., 1982; Huang
et al., 1985) and suggests that proteolysis does not regulate
the membrane fusion events of the guinea pig sperm acrosome reaction. The change from random hybrid membrane
tubules to vesicles did not occur in the principal segment
in the presence of serine protease inhibitors, but this may
have been due to the undispersed matrix rather than a direct
effect of the inhibitors on fusion.
However, in discussing the lack of effect of inhibitors
on fusion, other explanations should also be considered.
Firstly, the inhibitors may have had restricted access to
intracellular proteases. With the exception of SBTI (Mr
20,000), the inhibitors used in this study had a Mr < 500
and should have diffused rapidly into the acrosome, either
before addition of calcium or immediately upon initiation
of fusion. Secondly, the range of protease inhibitors may
have been inadequate. Mammalian spermatozoa are known
to contain a variety of proteases, the best-studied being the
proacrosin/acrosin system (Harrison, 1983). Unfortunately,
the subcellular distribution of many of these proteases is
unclear. Guinea pig sperm contain proacrosin/acrosin, two
other trypsin-like proteases (Arboleda and Gerton, 1987)
and dipeptidyl peptidase II (Talbot and DiCarlantonio,
1985; Hardy et al., 1991). Metalloendoproteases have been
reported in porcine, human and hamster spermatozoa (Gottleib and Meizel, 1987), and acid and alkaline proteases
may also be localized in the acrosome (Polakoski et al.,
1973; Ninjoor and Srivastava, 1985). The inhibitors used
in this study would have inhibited serine proteases
(acrosin), metalloendoproteases, acid proteases, thiol proteases, cathepsins and exopeptidases. Hence, while it is
possible that an unrecognised protease might be involved
in membrane fusion, we believe that most types of proteases were covered in this study. Thirdly, lysolecithin
Role of proteases in sperm acrosome reaction
169
Fig. 7. (A) Sperm induced to acrosome-react in the presence of NPGB and fixed with glutaraldehyde and tannic acid. Fusion has occurred
throughout the apical (as) and principal (ps) segments. In the apical segment, a thick layer of matrix (**) remains closely attached to the
hybrid membrane vesicles (v) on the ventral surface, while a thinner and less electron-dense layer of matrix (*) is present in the dorsal
region along with electron-dense matrix foci (arrows). es, equatorial segment; jz, junctional zone; s, hybrid membrane sheet. (B) and (C)
Sperm induced to acrosome-react in the presence of SBTI and fixed with glutaraldehyde and tannic acid. (B) Undispersed matrix (arrows)
links the hybrid membrane tubules (t) to the inner acrosomal membrane (im) in the principal segment. (C) There is a close association
between the undispersed matrix layer (**) on the ventral surface of the apical segment and the outer acrosomal membrane (om).
Accumulations of electron-dense material are located on the outer acrosomal membrane (arrows). Bars, 1 µm (A), 0.25 µm (B, C).
(LPC) might mediate fusion via a mechanism that does not
involve proteolysis and which is therefore unrepresentative
of the physiological acrosome reaction. Dravland et al.
(1984) reported that trypsin inhibitors prevented membrane
fusion during the hamster sperm acrosome reaction except
when the acrosome reaction was stimulated with LPC. We
chose to preincubate sperm with LPC because this method
synchronously acrosome-reacts a high percentage of cells
without affecting their viability or fertilizing ability (Fleming and Yanagimachi, 1981) and we have been unable to
consistently induce the acrosome reaction by overnight
incubation in calcium-deficient medium followed by the
addition of calcium (Yanagimachi and Usui, 1974). It
should also be noted that the structural pattern of fusions
described by Flaherty and Olson (1988, 1991) for the LPCmediated acrosome reaction can also be seen in the micrographs of Yanagimachi and Usui (1974) and Green
(1978b), who used overnight incubation and the Ca2+
ionophore A23187, respectively. Hence, the structural pattern of fusions is not due to the use of LPC but is a characteristic feature of the guinea pig sperm acrosome reaction.
Alternative hypotheses are therefore needed to explain
the structural pattern of fusion that occurs during the guinea
pig sperm acrosome reaction. One hypothesis is that the
pattern of membrane fusions is a reflection of the inherent
170
S. P. Flaherty and N. J. Swann
properties and heterogeneity of the outer acrosomal membrane and plasma membrane. Sperm membranes consist of
regionalized domains differing in composition and function
(Friend, 1982; Primakoff and Myles, 1983; Peterson et al.,
1987). The diffusion of proteins and lipids is restricted in
some domains and unrestricted in others (Cowan et al.,
1987; Wolf et al., 1988), and the composition of these
domains has been shown to change during capacitation
(Bearer and Friend, 1982; Stojanoff et al., 1988). Hence,
the specific structural manifestations of fusion during the
guinea pig sperm acrosome reaction may be regulated by
the organization of the plasma membrane and outer acrosomal membrane into domains of different composition and
fusibility. Phospholipase A2 activity has been detected in
plasma membranes and outer acrosomal membranes of
guinea pig sperm (Garcia et al., 1991), so a localized effect
of this enzyme on membrane lipids in specific membrane
domains might produce localized concentrations of
fusigenic lipids in those domains (Fleming and Yanagimachi, 1984). Cytoskeletal elements associated with the
outer acrosomal membrane might represent an alternative
or supplementary regulatory mechanism. An electron-dense
layer called the acrosomal lamina (Olson et al., 1989) is
present on the luminal surface of the outer acrosomal membrane in guinea pig, bull and hamster sperm and is probably a structural component of all eutherian sperm (Olson
and Winfrey, 1985b; Olson et al., 1985, 1987). In guinea
pig sperm, the acrosomal lamina has a filamentous substructure on the dorsal surface of the apical segment, and
these filaments impart a directional component to membrane fusion in this region (Flaherty and Olson, 1988; Olson
et al., 1987, 1989). Hence, regional differences in the composition of the acrosomal lamina or its interaction with the
membrane might regulate membrane fusion in different
regions of the acrosome.
The molecular mechanisms of membrane fusion are still
poorly understood, even in well-characterized systems such
as adrenal chromaffin cells (Plattner, 1989; Burgoyne,
1991). Recent work indicates that specific fusion proteins
(Satir et al., 1989; Stegmann et al., 1989) or differential
phosphorylation/dephosphorylation of membrane proteins
(Plattner, 1989) might be key events in the mechanism of
fusion. Such mechanisms might also operate during the
acrosome reaction and therefore represent additional
hypotheses to explain the unique pattern of fusions
observed during the guinea pig sperm acrosome reaction.
Our results using two different metalloendoprotease
inhibitors failed to provide any evidence of a role for these
enzymes in the membrane fusion events of the guinea pig
sperm acrosome reaction. This confirms results with human
sperm (Diaz-Perez et al., 1988) but is in contrast to the situation in the sea urchin (Farach et al., 1987). It should be
noted, however, that metalloendoprotease inhibitors have
also been shown to prevent a rise in intracellular calcium
(Burgoyne, 1991), so the results of Farach et al. (1987)
may be due to an effect on calcium transport rather than
a direct effect on membrane fusion. A recent study also
showed that trypsin inhibitors prevent the progesteronestimulated acrosome reaction of human sperm by directly
or indirectly interfering with calcium transport (Pillai and
Meizel, 1991).
Acrosomal matrix domains and interaction of the
matrix and outer acrosomal membrane
The acrosomal matrix is compartmentalized into zones of
differing electron density, some of which exhibit a crystalline substructure (Fawcett and Hollenberg, 1963;
Phillips, 1972; Olson and Winfrey, 1985a,b; Olson et al.,
1988). Recent studies have shown the acrosomal matrix to
consist of both enzymes and stable structural components,
and the structural components from hamster and guinea pig
sperm acrosomes have now been isolated and characterized
(Huang et al., 1985; Olson et al., 1988). Acrosomal proteases are sequestered in specific regions of the acrosomal
matrix; some are in the soluble fraction while others are
associated with structural components. This spacial sequestration of enzymes may regulate their differential release
during the acrosome reaction (Huang et al., 1985; Talbot
and DiCarlantonio, 1985; Olson et al., 1988; Noland et al.,
1989; Hardy et al., 1991).
The results of this study suggest that metalloendoproteases, acid proteases and exopeptidases play no role in dispersal of the acrosomal matrix during the acrosome reaction, whereas serine protease activity is required for
complete matrix dispersal. Other studies have shown that
serine protease inhibitors prevent matrix dispersal (Green,
1978a; Fraser, 1982; Perreault et al., 1982; Huang et al.,
1985). Two distinct layers of matrix and a group of electron-dense matrix foci in the apical segment, and an indistinct layer of matrix in the principal segment did not disperse when the acrosome reaction was induced in the
presence of serine protease inhibitors. The two matrix layers
in the apical segment correspond to components which have
been shown to contain proacrosin/acrosin activity (Huang
et al., 1985; Noland et al., 1989; Hardy et al., 1991). The
larger and more electron-dense of the two resistant matrix
layers was located in the ventral (concave) region of the
apical segment, and we noted a close association between
this layer and the outer acrosomal membrane or hybrid
membrane complex (Flaherty and Olson, 1991; present
study). Green (1978c) showed that this layer does not dissociate from the outer acrosomal membrane under hypotonic conditions even though much of the apical segment
matrix has cavitated. A similar association between the
outer acrosomal membrane and a specific structural component of the acrosomal matrix has been described for hamster sperm (Olson and Winfrey, 1985b; Olson et al., 1988).
The nature of the electron-dense matrix foci attached to the
dorsal layer in the apical segment is unknown.
In conclusion, we have used specific inhibitors to test the
hypothesis that proteases regulate the unique structural pattern of membrane fusions that occurs during the guinea pig
sperm acrosome reaction. While serine proteases (presumably acrosin) were required for dispersal of the acrosomal
matrix, our results indicate that the temporal and structural
pattern of membrane fusions is not regulated by proteases
in the lysolecithin-mediated acrosome reaction.
We thank Mark Crawford of TGAL, Matt Makinson for help
with the preparation of photographs, Ken Porter and the staff of
the Animal House at The Queen Elizabeth Hospital, Jim Wang
for help with data presentation, the Electron Microscope Unit at
The Queen Elizabeth Hospital and The Centre for Electron
Role of proteases in sperm acrosome reaction
Microscopy and Microstructure Analysis at The University of
Adelaide. This study was supported by a grant from the Australian
Research Council to S.F.
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(Received 10 July 1992 - Accepted 1 October 1992)