Biochemical Society Transactions
Diacylglycerol in the exocytosis of the mammalian sperm acrosome
E. R. S. Roldan* and R. A. P. Harrison
Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge
CB2 4AT, U.K.
284
Introduction
At fertilization, Ca’ -dependent exocytosis of the
sperm acrosomal granule (the so-called ‘acrosome
reaction’) exposes or releases hydrolytic enzymes
that allow the sperm cell to penetrate the oocyte
vestments. The process also primes the sperm head
plasma membrane for fusion with the oocyte itself.
As an example of exocytosis, the acrosome reaction
displays a number of unusual features: (1) the acrosome is large and asymmetric, covering and enclosing the anterior portion of the condensed nucleus;
(2) during the lifetime of the spermatozoon, the
outer acrosomal membrane lies in close apposition
to the overlying plasma membrane with which it
fuses during exocytosis; ( 3 ) the area over which the
plasma and outer acrosomal membranes fuse is
very large, with fusion taking place at numerous
sites and/or along arborescent pathways and (4) the
exocytosis process is very slow; many minutes can
elapse between triggering and membrane fusion,
even under physiological conditions.
Other features of the sperm system also
deserve comment. The spermatozoon is a very
asymmetric cell with large amounts of highly
domained external and internal membranes; moreover, the flagellar section with its mitochondria1 and
axonemal systems constitutes a clearly separate
compartment from the head section with the
nucleus and acrosomal granule. The biochemical
processes of the sperm cell are therefore widely dispersed and their locations difficult to define;
metabolites produced in one part of the cell may not
be available to other parts.
T o study the molecular events in the sperm
acrosome reaction, we have used a model in which
exocytosis is triggered by treatment with the calcium ionophore A23187 [ l , 21. W e were able to
show early in our studies that at least three Ca2+dependent events are involved in the sequence leading to membrane fusion; the early events require
only micromolar levels of the ion, whereas later
events require millimolar levels; it appears that such
Ca2+ must be supplied to the interior of the cell
+
~
Abbreviations used: DAG, diacylglycerol; LAT, lysophosphatide-acyltransferase; PIJA2, phospholipase A2;
I’PI, polyphosphoinositide.
*Towhom correspondence should be addressed.
Volume 21
from the external milieu. These findings have provided us with a means of analysing the sequence
from which we have developed a working hypothesis of the events involved. Our data suggest that
diacylglycerol (DAG) plays an unusual key role.
Generation of DAG
Stimulation of mammalian or invertebrate
spermatozoa either with natural agonists or with
A23187 and CaZ+results in a considerable breakdown of the polyphosphoinositides (PPIs) [ 3-51 by
phosphoinositidase C (identified in human [6] and
bull spermatozoa [7]). In ram spermatozoa treated
with A23187/Ca2+ the breakdown is very rapid
and seems essential for exocytosis [3]. PPI hydrolysis is accompanied by a concomitant generation
of DAG [8] and inositol 1,4,5-trisphosphate [9] but,
although both compounds peak at about the same
time that PPI breakdown is completed (that is, 3
min), the DAG levels remain high for some 10 min
before declining slowly.
Quantitation of the DAG mass generated after
ionophore stimulation has revealed that the amount
is too great to have been derived entirely from the
PPIs [8]. It appears therefore that there are mechanisms capable of generating DAG from other sources
as well. Mammalian spermatozoa have a Ca2 dependent phosphatidylcholine-specific phospholipase C [lo], but there is no indication as yet that
sperm stimulation results in activation of this
enzyme. It is also not known whether mammalian
sperm have a phospholipase D, although stimulation
of sea urchin spermatozoa with a natural ligand
(fucose-sulphate glycoconjugate) has been shown to
activate a Ca2+-dependent-phospholipase D,
generating phosphatidate and DAG [ 111.
Our preliminary studies suggest that generation of DAG from other sources takes place concomitantly with that from PPIs, hence it appears
that all the pathways are activated together rather
than in sequence. Stimulation of spermatozoa under
conditions that activate PPI breakdown also results
in maximal DAG mass accumulation, whereas there
is no DAG mass rise with treatment conditions that
do not lead to PPI breakdown (Table 1). It remains
to be seen whether PPI-derived DAG may exert a
positive feedback effect activating phosphatidylcholine-specific phospholipase C and/or phospho+
Phospholipid Translocation, Asymmetry and Membrane Fusion
Table I
Changes in polyphosphoinositides and diacylglycerol after treatment of ram
spermatozoa with A23 I87 and bivalent cations
Spermatozoa were labelled with 250 p C i [32P]P,/mIfor 45 min and then treated with
I p M A23 I87 in the absence or in the presence of bivalent cations (3 mM) or I mM
EGTA. After 5 min. the samples were precipitated with perchloric acid and lipids were
extracted with chloroform/methanol/concentrated HCI and separated by t.1.c. as
described [3]. For quantitation of DAG, parallel unlabelled samples were treated similarly
and lipids were extracted with chloroform/methanol, separated by t.1.c. and the mass was
quantified by Coomassie Blue staining and densitometry using I ,2-dioleoyl-sn-glycerolas
a standard [16]. Results are the means (k5E.M.) from three experiments. *Compared
with control: P < 0.01; +compared with A23 I 87/Ca2+:P < 0.01.
PPI (c.p.m./l O8 spermatozoa)
285
DAG
(Pugl I O8
Treatment
Control
A23 I 87/Ca2
+
A23 I87 alone
A23 I 87lEGTA
A23 I 87/Mg2
+
[32 PIPtdIn s4P
[32P]Ptdlns(4,5)P,
spermatozoa)
55185f331 I
I0485 f2759"
I 5 I 07 f 2704"
56289 f 23 l 8 t
6898 I f 6622t
3471Of2776
8851 f728"
9718f 1915"
38875 f97 I t
3 I239 f2082t
I .4 f0.05
lipase I ) and thus leading to higher DAG levels, as
in some somatic cells [ 121.
Sperm membranes contain high levels of
unusual polyunsaturated fatty-acyl residues associated with a considerable variety of diacyl-, alkylacyl- and alkenylacylphosphoglycerides [ 13, 141.
Thus numerous molecular species of diglyceride
may be generated at sperm activation that, by a
combination of molecular specificity and compartmental restriction, might achieve accurate targeting
of messenger action. It is noteworthy that 'resting'
levels of DAGs in spermatozoa seem very high [Z,
81; one possible explanation for this feature is that
these DAGs are located in compartments other than
the one involved in exocytosis; alternatively they
may have no role because of their structure.
Catabolism of DAG
DAG can be catabolized via two pathways: phosphorylation to phosphatidate by DAG kinase (the
more active pathway in most cells) or deacylation
by DAG lipase which removes a fatty acid to generate monoacylglycerol. Since PPI-derived DAG
usually contains arachidonate in position 2 [ 1.51, the
latter pathway can constitute a way of releasing
arachidonate for eicosanoid synthesis in certain cell
types [15]. Using inhibitors of DAG kinase and
DAG lipase and quantifying metabolites after cell
stimulation, we have been able to demonstrate that
the DAG generated by ionophore treatment can be
3.5 f 0.09"
3.5 f0. 12"
I .5f0.08t
I .6 f0.07t
catabolized via both pathways in spermatozoa [ 161.
However, inhibition of DAG catabolism via either
route, with consequent increase in net DAG mass,
leads to an increased rate of exocytosis [ 161; addition of exogenous phosphatidate on the other hand
does not enhance exocytosis. W e believe therefore
that the DAG released during sperm activation
plays a direct role rather than serving as a source of
other active metabolites with further roles in exocytosis.
Further evidence for the involvement
of DAG in acrosomal exocytosis
As an alternative to using catabolic inhibitors, the
levels of DAG can be increased in stimulated
spermatozoa by adding exogenous LIAGs. This
treatment also leads to enhanced rates of exocytosis,
whereas treatment with exogenous DAGs is without
effect in the absence of A23 187/CaL+ stimulation
[ 8, 161. If neomycin is used to block PPI breakdown
and to inhibit subsequent exocytosis [ 31, addition of
exogenous DAG will restore exocytosis to control
levels [16]. This suggests strongly that the site of
action of DAG is 'downstream' of PPI breakdown.
Role of DAG
Since DAG does not seem to serve as a substrate to
generate other active metabolites, various alternative
roles must be considered.
I993
Biochemical Society Transactions
286
DAG could be acting directly as a fusogen
[17]. However this seems improbable for two
reasons. Firstly DAG accumulation peaks very
rapidly (about 3 min) whereas exocytosis begins to
occur in the cell population several minutes later
and continues for over 30 min [8, 161. Secondly we
have detected at least two Ca'+-requiring events
that take place downstream of DAG production,
both of which are essential for exocytosis [3]. It
seems likely therefore that DAG is exerting a
second messenger effect.
The most commonly recognized messenger
role for DAG in somatic cells is the activation of
protein kinase C. In spermatozoa our own studies
[18] indicate that, during the course of A231871
Ca"-induced exocytosis, treatment with phorbol
esters neither modifies protein phosphorylation nor
affects the time course of exocytosis; moreover, we
have been unable to detect specific binding of
phorbol esters to the cells (an indication of the
presence of active protein kinase C). Work by
others has resulted in paradoxical data regarding
the effects of phorbol esters on acrosomal exocytosis; for discussion see [ 1, 21. For example, addition
of phorbol esters to mouse spermatozoa induced to
undergo exocytosis with the natural agonist, stimulated the initial phases of exocytosis, but retarded
later stages [ 191; in addition, the reversible effects
conflicted with the known protracted stimulation of
these compounds on other cells. Recent studies
have detected protein kinase C activity in partially
purified sperm preparations [20,21], but there is no
indication as to which cell compartment contained
the activity. Moreover, immunocytochemical
analyses have revealed the presence of three protein
kinase C isozymes (a,/3, y ) in bull, ram and human
spermatozoa [20-221, but their locations seem to be
restricted to cell domains not involved in exocytosis. Finally, our investigations of the effects of
exogenous DAGs on spermatozoa have shown that
both 1, 2- and lJ-isomers enhance exocytosis [ 161,
although only the former is known to activate protein kinase C [23].
Since protein kinase C does not appear to be
involved in events taking place downstream of Ca'+
entry, we have suggested [ 1, 21 that messenger
effect of DAG in spermatozoa may be directed at a
different cellular target, namely phospholipase A?
(PI,A2), and we have sought evidence in favour of
this hypothesis.
The importance of PI,AL activation during
acrosomal exocytosis relates to the ability of the
enzyme to generate both arachidonic (and other
fatty) acids and lysophospholipids, through hydro-
Volume 21
lysis of membrane phospholipids. These metabolites have been shown to be involved in membrane
perturbation and fusion [24, 251. The characterization (for example [26-281) and localization
(for example [29]) of mammalian sperm PLA, has
received considerable attention. In spite of these
efforts, the evidence for a role of PLA, during acrosoma1 exocytosis has remained largely circumstantial and has relied exclusively on the response of
sperm cells to putative PLA2 inhibitors [30, 311 or
to exogenously added PI,A,-derived metabolites
[32-34]. Preliminary evidence has been presented
for an association between PLALactivity (measured
as an increase in labelled arachidonate or lysophospholipids) and acrosomal exocytosis in human [ 35 1
and in hamster spermatozoa [ 361.
Recent work has demonstrated that DAGs
can stimulate the Calf-dependent PI,A2 in in vitro
assays of ram-sperm sonicates [27]. The effect was
seen with both 1,2- and IJ-DAGs, suggesting that
the diglycerides may be able to stimulate enzyme
activity directly [37] or by affecting substrate
presentation [ 381. Further work has shown that
alkylacylglycerols are capable also of stimulating
enzyme activity under these conditions and, moreover, that extracts from sperm cells stimulated with
A23 187/Ca'+ and either diacyl- or alkylacylglycerol showed higher levels of PI,A? activity than
did control extracts (E. R. S. Roldan, unpublished
results).
A subsequent study has provided evidence for
the stimulation of PLA? by DAGs, by measuring
release of [ "C]arachidonate from phospholipids in
pre-labelled spermatozoa. 1,evels of arachidonate
and lysophospholipids in intact cells are regulated
by the interaction between PI,AI and lysophosphatide-acyltransferase (LAT), the former hydrolysing
phospholipids, the latter reacylating the resulting
lysocompounds to regenerate the phospholipid
pools. Therefore it should be noted that the effects
of DAGs on PLA, [39] can in fact occur in parallel
with the effects of DAG on LAT 140, 411 and this
has to be taken into account when attempting to
understand regulation of arachidonate or lysophospholipid release in cells.
When
spermatozoa
pre-labelled
with
[ "Clarachidonate were treated with A23 187/CaL',
the levels of free arachidonate rose greatly, more
slowly than the levels of DAG but in close parallel
with exocytosis. Pre-incubation of labelled spermatozoa with DAG before treatment with A23187/
Ca2+ resulted in higher levels of arachidonate
release than in cells treated with A23187/Ca"
alone and also resulted in an acceleration of exocy-
Phospholipid Translocation,Asymmetry and Membrane Fusion
Table 2
Effect of diacylglycerols on arachidonate release and acrosomal exocytosis
Ram spermatozoa were labelled with 0.5 pCi ['4C]arachidonate/mlfor 60 min, washed
and resuspended in a saline medium with 3 mM Ca2+.Labelled cells were incubated in
the absence or presence of DAGs for 10 min and were then exposed to either I pM
A23 187 or dimethylsulphoxide.After 10 min, incubations were stopped and lipids were
extracted and resolved by t.1.c.; the radioactivity in each lipid spot was quantified by
liquid-scintillation counting. Parallel unlabelled samples were treated similarly and the
incubations stopped at 10 min and examined for the occurrence of acrosomal exocytosis.
Results are the means (kS.E.M.) of duplicate assays from four experiments. *Compared
with control: P < 0.0 I ; +compared with A23 I 87/Ca2+:P < 0.05.
['4C]arachidonatereleased
after 10 min
(c.p.m./lO8 spermatozoa)
lOf3.5
45 f 4.2*
Il244f812t
62 f 5.3t
9787 f652t
4284 f 528"
65 k 5.6t
9f2.1
IOf3.2
+
+ I ,2-DAG (25 pM)
Acrosome reactions
after 10 min (%)
2200 f I54
6941 f731*
Control
A23 I 87/Ca2
+ I ,3-DAG (25 pM)
I ,2-DAG alone
I ,3-DAG alone
2354 f249
tosis. Both 1,2- and 1,3-DAG isomers were effective
in raising arachidonate levels, as well as accelerating
exocytosis (Table 2). Interestingly treatment with
1,2-DAG alone (in the absence of A23187/CaZ+,
resulted in a rise in arachidonate levels; we believe
this may be due to an inhibitory effect on LAT [40]
which, by preventing the re-acylation of lysophospholipids, leads to an accumulation of free arachidonate. (Although some PLA2 activity may be
taking place under these conditions, maximal
enzyme activity only occurs after Ca'+ internalization.) Higher arachidonate levels in cells exposed
only to 1,ZDAG did not result in exocytosis, in
accordance with the concept that membrane fusion
itself requires Ca'+ entry (see above).
W e still do not know what the role of the
metabolites generated via control of PLA2 and LAT
activities may be. Emphasis has been given to the
potential role of sperm lysophospholipids in membrane fusion during exocytosis [32],but less attention (see [34]) has been paid to the possible role of
unsaturated fatty acids in events related to membrane perturbation or to fusion after synexinmediated changes [25].
The sequence of events leading to
acrosomal exocytosis: A hypothesis
Based on earlier findings we believe that acrosomal
exocytosis involves at least three processes requiring Ca'+. Our current working hypothesis is as fol-
287
lows (Figure 1): Upon Ca2+ entry there is a
significant Ca' -dependent breakdown of the PPIs
to generate DAG, this probably constitutes the first
Ca'+-requiring step. DAG could derive also from
the action of other phospholipases activated concomitantly. The DAG messenger action seems to
relate to a direct activation of PLA2 (which may
constitute the second Ca'+-requiring event),
together with a possible parallel inhibition of LAT.
The metabolites generated through PLA, action
and enhanced by LAT inhibition (that is, acyl- or
alkyllysophospholipids and polyunsaturated fatty
acids) may act in a concerted way to cause membrane fusion, probably the third and final CaL+requiring event.
+
Conclusions
Our studies have provided evidence that diradylglycerols play an important messenger role in
amplifying the biochemical processes that lead to
membrane fusion during the sperm acrosome reaction. It is certain that the diradylglycerols are
derived from the Ca' -dependent PPI breakdown
that is a very early event following Ca2+ entry, but
they may also be derived from the action of other
phospholipases. Rather than targeting protein kinse
C to control enzyme action through phosphorylation, the diradylglycerols appear to act by directly
stimulating PLA, to bring about an increase in
levels of fusogenic free fatty acids and lysophospha+
I993
Biochemical Society Transactions
Figure I
Possible mechanisms leading to membrane fusion during exocytosis of the
sperm acrosome
Abbreviations used: AA, arachidonic acid; FFA, free fatty acidPC, phosphatidylcholine;
PC-PLC, phosphatidylcholine-specific phospholipase C; PIC, phosphoinositidase C; PLD,
phospholipase I2 R, receptor.
288
lysoPC
+
FFA (AA)
cyclooxyganase/
lipoxygenasc
pathways
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Fertilization in Mammals (Havister, H. D.?
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tides; the build-up of these products may also be
engendered by a concomitant inhibition of LAT.
Much further work is needed to define the sources
of the diradylglycerols, the particular species
released and their effector abilities on target
enzymes. But, given the unusual composition of
sperm phosphoglycerides and the highly compartmentalized nature of the sperm cell, it seems likely
that a high degree of specificity of composition and
location of action will be revealed.
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Received 16 December 1992
Protein kinase C, membrane fusion and platelet granule secretion
Jon M. Gerrard”, Archibald McNicol and Satya P. Saxena
Manitoba Institute of Cell Biology, 100 Olivia Street, Winnipeg, Manitoba, R3E OV9, Canada
Introduction
ATP was depleted sufficiently [Z]. At approximately
The study of granule secretion in blood platelets
began with the work of Grette, w h o first coined the
term ‘release reaction’ t o describe the secretion of
granule contents [l]. A number of years later
Holmsen established clearly that this process
the same time, White was studying electronmicroscopic changes associated with secretion and
noted that platelets were unusual because, following
activation, their granules moved initially towards the
centre of the platelet rather than towards the
periphery [3]. White demonstrated that a n
actin-myosin network surrounded the granules
associated with secretion, which appeared t o
squeeze the granules towards the centre of the cell
a n d might have a role in facilitating the membrane
required energy a n d would not occur if cellular
Abbreviation used: I’MA, phorhol 12-myristate 13acetate.
*Towhom correspondence should be addressed.
I993
289