BIOLOGY
REPRODUCTION
OF
Role
of Human
47,
75 1-759
Sperm
Phospholipase
(1992)
Phospholipase
A2 Activity M.R.
Department
on Membrane
FRY,
S.S.
GHOSH,
and
Molecular Richmond,
of Biochemistry
Reproductive
A2 in Fertilization:
Evaluation,3
Roche
Effects
Perturbations
J.M.
EAST,3
and
and
R.C.
Oocyte
Inhibitor
of
Penetration’
FRANSON2
Biophysics, Virginia Virginia 23298
Biomedical
of a Novel
Laboratoiy,
Commonwealth Richmond,
University
Virginia
23229
events
of fertilization
ABSTRACT Phospholipase examined.
A, was
Highly pH
alkaline
pendent
with
istration
5 mM
Cad,
80%
(±
10%)
had
little
to no
may
contribute
effect
on
enzyme
inhibited sperm the
was
Mammalian
fusion
phospholipases
acrosome events
preincubated
sophospholipids.
Both
phospholipid
bilayer
[1]. Several membrane dogenous the sperm sion
and
events fusion
reaction.
These
in mammalian
ubiquitous
act
vesiculation
reported as endogenous
sperm
plasma
acid is released from the calcium ionophore
pholipase A2 have also been proposed and oocyte plasma membranes [12]
‘This
work
was supported
23298-0614.
Box
614,
by National
B
that
Sperm
sperm
of cortical
Station,
Virginia
Dept.
Commonwealth
of Health
grant
University,
sperm
by
approx-
resonance
phospholipase
energy
admin-
transfer.
degradation of zona
A, and
granule
with
at neutral bromide,
and
were pellucidaB,,
modulators
its
from
A2 activity
has
fertilized
mouse are
been
oocytes
characterized
[3], and human calcium-dependent,
by mepacrine.
4-bromophenacyl
bromide
Inhibitors
such
have
used
to probe the role of phospholipase reaction [3-5, 16-18]. However, most
described
to
date
lack
specificity
evidence A2. In the
membranes,
affect
and
therefore
been
A2 in the inhibitors have
not
to support or exclude a role present studies we have used
a novel enzyme-targeted inhibitor human nonpancreatic phospholipase study the ability of human sperm turb
[4]
pH, sensitive to alkylation by and (except for the human sperm
extensively acrosome
provided convincing for phospholipase
ac-
vesicle
of in vitro A2 activity phospholipase fusion,
and
and in situ [19,201 to A2 to per-
participate
in
fertilization. We demonstrate that prostaglandin B5 (PGBX), an inhibitor of in vitro human sperm phospholipase A2 activity, blocks vesicle fusion in vitro, enzyme-induced inflammation in vivo, and human sperm penetration of zona-free oocytes. Inhibition of penetration was not mediated by in-
[7, 8] the phos-
hibition
of the
sperm
acrosome
AND
reaction.
METHODS
Materials
DK 42615. and Molecular Richmond,
contents
A2) inhibited
as mepacrine
of endogenous and cis-un-
of Biochemistry
Human
chloride
penetration
phospholipase
phospholipase
event, fu-
in the fusion of sperm and the exocytotic re-
Institutes
C. Franson,
bromide. calcium
phospholipid
MATERIALS
Dr. Richard MCV
and
and
by oral
were incubated with prostaglandin B,, (IC,,., Capacitation in the presence of prostag)andin
suggest
June 24, 1992. June 10, 1991.
‘Correspondence: Biophysics,
prostaglandin
optimally active 4-bromophenacyl
involve of en-
human
saturated fatty acids induce vesicle-membrane fusion and, in addition to platelet-activating factor, stimulate acrosome reaction in vitro [9-111. Similar roles for
Accepted Received
of fluorescence fusion
A, in vitro
inhibited
in hamster [141, guinea pig [151, sperm. In general, these enzymes
the
membrane
rosome reaction, demonstrating activation phospholipase A2 activity [6]. Lysophospholipids
1 mM
and
In vitro
fusogens
prelabeled A23187-induced
of
to
in a dose-de-
[13].
the underlying acrosomal membrane [2]. While the acrosome reaction has not been well characterized at the molecular level, sperm acrosomal proteins including phospholipase A2 are thought to be involved in the process [36]. Arachidonic sperm during
was
4-bromophenacyl
efficiency both
inhibited
phospholipases that
was
at neutral
fertilization.
to perturb
A2 activity. The first such reaction, involves the organized of the
5 M
results
enzymes
in mammalian fertilization that can be modulated by activation
phospholipase acrosome
and
are
may
with
optimally
was
human edema
presence
and
with
to generate predominantly cisthe sn-2 position and 1-acyl ly-
products
to inhibit
in the
hydrolysis,
coli
Activity
enzyme
lease
A2 are
phospholipid fatty acids from
reported
(o.D.)
turbidity
fusion
Escbericbia
a dose-dependent of the
INTRODUCTION that hydrolyze unsaturated
M)
vesicles
phospholipid
membrane
moI/min/mg).
in a dose-dependent fashion when sperm motility was not affected by this treatment.
in vitro
to membrane
= 20
or by pretreatment
by
in the
1-’4C)oleate-labeled
induced
of phosphatidylserine
accompanied
when
1.5
=
foot pad
role
of[
activity
B1 (IC50,
by monitoring
was
60%
free hamster oocytes was 15 JLM) during capacitation;
(specific
mouse
10 mg/kg)
as determined
than
NaCI
into
potential
its
phospholipids
the
mM
fusion
and
sperm
of prostaglandin
induced
fusion
by more
150
A2 injected
jtg)
enzyme-induced
inhibited
and
B,, (IC50
A, (10
human
hydrolyzed
by an oligomer
of prostaglandin
imately
from
enzyme
phospholipase
Sperm
phospholipase This
purified
manner
in situ.
isolated
PGB5 was synthesized, nated by Drs. George
VA
FAR: 804-786-1473.
751
characterized, L. Nelson (Saint
and generously doJoseph’s University,
FRY
752
Philadelphia,
PA) and
ical
Philadelphia,
College,
Thomas
M. Devlin PA),
and
(Hahnemann
the
Office
Med-
of Naval
Re-
ET AL.
less
otherwise
phospholipase
5 ng
E. co/i
partially
purified
phospholipid
sperm (9.8
X 10
search (Bethesda, MD). PGBX, the polymeric derivative first synthesized by Polis et a!. [211 has an average molecular weight of 2400. Female golden hamsters (6-12 wk old) and
cells, buffer
male River
dissolved in DMSO or used as a water-soluble sodium salt. DMSO (5%, v/v) has little or no effect on control enzyme
tion
albino mice (CD-i strain) were obtained from Charles Breeding Laboratories (Wilmington, MA). BSA (fraceCG,
V),
hCG,
hyaluronidase,
sin, polyvinylpyrrolidine-40, thiocyanate-conjugated calcium ionophore mophenacyl bromide
bovine propidium
Pium A23187, were
pancreatic
iodide, agglutinin
sa&’um prostaglandin purchased
from
ical Co. (St. Louis, MO). Phosphatidylserine (PS, sodium salt), N-4-nitrobenzo-2-oxaphatidylethanolamine (NBD-PE), mine B sulfonyl)-phosphatidylethanolamine purchased
from
Avanti
tryp-
4-broChem-
N-(lissamine (Rho-PE)
Biochemicals
sham,
UK).
All other
analytical
grade.
Isolation
of Human
reagents
and
chemicals
([1(Amer-
used
were
per
Pooled
human
semen
A2
generously
provided
was
plicate
and
Laboratories diluted 1:1
containing X g for
25 mM sucrose (buffer A) and centrifuged 15 mm to sediment cells. The pellet was
times
with
(Richmond, with 50 mM
buffer
A, and
VA). The fluid (50-100 Tris-HCI buffer (pH 7.5)
each
successive
at 400 washed
wash
con-
tained diminishing levels of phospholipase A2 activity. Washing removed 16% of the total activity, indicating that some enzyme is loosely associated with sperm membranes. To solubilize the phospholipase A2 activity, the washed cells were resuspended in 50 mM Tris-HCI buffer (pH 7.5) and disrupted with three bursts (30 sec each) from a Branson probe sonicator. The sonicate was then mixed with an equal volume of ice-cold 0.36 N sulfuric acid, stirred overnight at 4#{176}C, and supematant
centrifuged at 27 000 was dialyzed against
4.5), 0.5 M NaCI and centrifuged tion enriched in phospholipase was applied to a sulfopropyl-Sephadex umn in 0.5 1.0 M NaCI.
M NaCI and The fractions
tivity were pooled tion (Amicon YM5
The resulting sodium acetate (pH
10 mM to yield
a supernatant fracThis acid extract cation exchange col-
A2 activity.
activity was eluted phospholipase
with A2 ac-
and concentrated 12-fold by ultrafiltrafilter) in the presence of 0.005% Triton fraction, purified = 20 p.mol/min/mg),
mg
mean
corrected
mixtures
stopped
in all of two
were
by the
ad-
(1/2, v/v). Total lipids of Bligh and Dyer [23], sepquantitated described
by [22].
of Bradford p.mol fatty
[24]. acid
by the method is expressed
as
or as percent
of control.
of two
for
experiments
background
experiments)
All data
done
hydrolysis
in du-
(less
than
repeated.
of Edema
approxiwas
A2 Assay
Phospholipase A2 activity was measured using [1-’4C]oleatelabeled, autoclaved Eccheridna co/i as substrate [22]. Reaction mixtures in a total volume of 0.5 ml contained (un-
phospholipase
A2, in a total
10 mM sodium acetate was injected into the mice tion
weighing the mice
moved
at the
for
percent
volume
20-25 g. Forty-five minutes were killed, and both hind ankle
edema
of 50 .d
(pH 4.5) containing foot pad of the hind
joint
calculated as the weight weight of the nontreated
and
weighed.
after limbs
the injecwere re-
Percent
edema
was
of the injected limb divided by the limb times 100 and was corrected
in the
sham-injected
(isotonic
saline)
(104 ± 3%). PGBX was administered by gastric 60 mm prior to the injection of the enzyme. analysis of the data was performed by analysis
variance of two
experiments
Alkylation ume
Edema (n =
(ANOVA).
of Phosphol4xise
is expressed 6).
mM
NaCI,
solved acyl
and
Triton
A2 (40 p.g), mM Tris-HC1
X-100
(w/v),
reaction
mophenacyl phospholipase
or enzyme conditions.
alone
were
Aliquots
bromide-treated A2 activity
mean
SD
±
ability
vol100
incubated
for
bromide 4-bromophen-
dis-
incubated of controls
enzyme and
in a total (pH 8.0),
was
at 37#{176}C with 320 M 4-bromophenacyl in DMSO. Controls containing either
bromide
same
0.02%
as the
inStaof
A2
Human sperm phospholipase of 1.0 ml containing 30
90 mm
of
50 mM NaCI, limb of male
control tubation tistical
g for 15 mm.
enzymic containing
X-100 (w/v). The concentrated mately 25-fold (specific activity used in all studies.
Phosphohpase
X
per
the
was
PGBX
of
by Roche
Biomedical ml) was
four
mm
are
Sperm
Phospholiase
reaction
at 37#{176}C and
determined A2 activity
Measurement Sperm
while
by thin-layer chromatography, and scintillation spectroscopy as previously
presented 1%
(DMSO),
otherwise,
10 minutes
of 3 ml chloroform/methanol extracted by the method
Protein was Phospholipase 1-
sulfoxide
indicated
for
released
AL);
palmitoyl,24 i-’4C]linoleoyl-3-phosphatidylethanolamine 14C]-PE) was obtained from Amersham International
in dimethyl
Unless
arated liquid
rhodawere
(Birmingham,
activity. dition were
from bovine brain 1 ,3-diazole-phos-
and
dissolved
cpm), 5 mM calcium chloride, 50 mM Tris-HCI 7.5), and 150 mM NaC1. Prostaglandin B1 was
incubated
fluoroiso(FITC-PSA),
B1, and Sigma
10000 (pH
indicated) A2, 9 nmol
were to induce
under and
the 4-bro-
assayed
for
edema
as
described.
Measurement Phospholipid in turbidity
of Vesicle vesicle (OD)
and
Fusion fusion was efficiency
monitored of transfer
by changes of fluores-
cence resonance energy [251. Fluorescent-labeled vesicles containing 25 pg total phospholipid (PS/NBD-PE/Rho-PE, 96/2/2, w/w/w) and vesicles (225 pg PS) containing [1-
ROLE
‘4C]linoleate-labeled [1 -14C]-PE/PS,
OF
SPERM
HUMAN
phosphatidylethanolamine w/w) were prepared
1/1500,
PHOSPHOLIPASE
(24 000 separately
cpm, by
sonicating (two 30-sec bursts) lipids in 10 mM Tris-HC1, pH 7.5, 150 mM NaC1 (buffer B). Reaction mixtures, in a total volume
of
beled were
3 ml,
vesicles incubated
contained
fluorescent-
(250 p.g total phospholipid) at 25#{176}C for 10 mm. Calcium
(5 or 10 M), and human sperm were added to reaction mixtures excited
at 480
fluorescence was measured
nm
(slit
emission using
photometer
(Kyob,
in the
width
Japan).
B and PGBX
a change
in
transfer
of fluorescence =
Xem
of divalent
480
=
(Xex
590
cations,
res-
nm, Xem = 530 nm) was obas excitation
at
480 nm resulted in fluorescence at 590 nm. The addition of 2.5 mM calcium chloride increased the fluorescence of NBD at 530 nm (from F530 = 0.5 to F5 = 6.8), which could be inhibited ning, Triton 0.03%,
by EDTA (F530 = 2, data not shown). After X-i00 was added to a final concentration
and
efficiency culated
emission (E)
at 530
nm
of resonance
was
(F0)
energy
scanof
determined.
tranfer
(RET)
was
[1
=
(F530/F0)]
-
a Perkin-Elmer
Lambda Hydrolysis
(Norwalk,
CT).
fusion measure
was assessed the release
cpm released is reported plicate determinations.
Oocyte
Assay
sperm
fertilizing
a modification tained with
of the
described from
healthy
resuspended of PGB5. The
donors
Whitten,
were
stored
the
were
and
at 37#{176}C in air
second
mean
of du-
measured
oocyte Briefly,
allowed
Whittingham
hamsters
injection;
using
penetration ejaculates
to liquefy, culture
pH 7.4-7.6 (B’W/BSA), 10 mm. The pellet was
injection of 25 IU of eCG 24 IU of hCG 54 h later. after
hamster
in BWW/BSA with sperm concentration
added. Female golden
was
et a!. [26].
sperm/mI and incubated capacitation. Aliquots (100 placed in a 35-mm culture and
the
associated
and
obdiluted
medium and centriwashed and
or without the was adjusted
sodium salt to 1 x io
at 37#{176}C for 18 h to allow in vitro i.l) of the sperm suspension were dish, covered with mineral oil, until were
zona-free
hamster
superovulated
followed by an i.p. The hamsters were the
cumuli
were
oocytes by an
i.p.
injection of killed 16 h isolated
washed
35-mm
with
BWW
culture
PGB5, and incubated were then washed to
and
examined
me-
dishes
confor 2 h remove
by phase-contrast
X magnification. Penetration containing at least one swollen
its associated
Acrosomal
±
sperm
microscopy at 400 fined by an oocyte’s
was desperm
tail.
Evaluation
Acrosomal
status
was
determined
by the
method
of Mi-
yazaki et al. [27], in which the binding of a fluoresceinated pea lectin to human sperm acrosomal cap was detected by flow cytometry. Human semen was obtained from a normal donor,
allowed
to liquefy
for concentration by centrifugation
and of
for 30 mm
motility. semen
Piscataway,
at 25#{176}C, and
Motile through
NJ) gradient
evaluated
sperm were a two-step
and
[28,29].
The
selected Percoll
sperm
were
of 1 x i07 sperm/mi and divided into 0.5-mi aliquots. (0-50 pM) was added as indicated and sperm were bated at 37#{176}C, 5% CO2 for 18 h to allow capacitation. cium
ionophore
A23187
(10
p.M in DMSO)
was
PGBX incuCal-
added
for
the final 1.5 h of capacitation as a positive control. DMSO (2 p.1, 0.4% total reaction volume) was added to all other samples.
Samples
dide, a dye that at 25#{176}C to stain
900
zona-free
containing 0.3% BSA, fuged at 300 x g for
0.5-mI aliquots to percent of total
are
potential
by Rogers
Biggers,
all data
sperm oocytes
were
incubated
x g for
with
5 mm.
Pellets
were
resuspended
1 ml
PBS,
fluorescence rosome-reacted
pH
7.4,
centrifuged
dard filters (515-530 655 nm for propidium ple, sperm were dium iodide to cence
nm for iodide
analyzed indicate
of five thousand
X g for
10 mm,
was
of acand
cytometry on an FL), using stan-
FITC fluorescence fluorescence). For
sperm
was
samples were were washed
segment fluorescence
by flow Hialeah,
for nuclear viability, and
viable
ml
7.4. FITC-PSA has been of intact sperm, while
appears only at the equatorial sperm [30]. Acrosomal analyzed (Coulter,
(FITC-PSA)
and Sperm
at 300
in 0.5 ml PBS, pH the acrosomal region
supravital staining were EPICS 753 flow cytometer
5 mm iodide
in 0.15
agglutinin
added to a final 20-p.g/ml concentration incubated for 15 mm at 25#{176}C in the dark. and resuspended shown to bind
io-
for
by adding 0.35 ml 100% of 70% ethanol. Samples mm in the dark. Fluoroiso-
P. satitin
thiocyanate-conjugated
by DNA, propidium
samples through 5 ml of 4% (PVP-40) in PBS, pH 7.4 at
PBS, pH 7.4 and permeabilized ethanol to a final concentration were incubated at 4#{176}C for 30
with
1 p.g propidium
fluoresces when bound dead sperm. Unbound
was removed by centrifuging (w/v) polyvinyl pyrrolidmne-40
Penetration
Human assay
and
taining capacitated at 37#{176}C in air. The
were
to the
cal-
4A UV/VIS Spectrophotometer of phospholipid during vesicle The
oocytes added
(Pharmacia,
x 100,
by lipid-extracting of [‘4C}lmnoleate.
zona-free
washed twice (300 x g, 10 mm), resuspended in B’W/ BSA, and re-evaluated for concentration and motility. Sample volume was diluted with BWW/BSA to a concentration
and is inversely related to the extent of vesicle fusion. Prior to the addition of Triton X-100, turbidity was monitored by measuring optical density of the vesicles at 400 nm (O.D.) on
The
immediately
sur0.1% pel-
The
as: E
lucidae.
head
753
FERTILIZATION
with 0.1% hya!uronidase for 10 mm to remove cells, and then the oocytes were treated with pancreatic trypsmn for 1 mm to remove zonae
loosely
A2 (10 g) Samples were and
treated rounding bovine dium,
(F530, slit width = 10 nm) RF-5000 spectrofluoro-
NBD-PE 530 nm,
=
absence
in buffer chloride,
5 nm),
=
The
[1-’4C-PE]-la-
phospholipase as indicated.
at 530 nm a Shimadzu
onance energy between nm) and Rho-PE (Xex served
and
A2 IN
exclusion acrosomal examined.
and each
635sam-
of propifluoresPercent
FRY
754
ET AL.
30
A
B
E C
20
E 0
E 10 > I-
C-)
0
I
I
I
I
4
6
8
10
0.01
0.10
pH
10.00
1.00
CaCI2
100.00
(mM)
FIG. 1. Optimal pH and calcium requirements for human sperm phospholipase A, activity. Panel A: Standard reaction mixtures contained 20 mM buffer l sodium acetate, pH 3.5-6.5; 0 Hepes. pH 6.5-7.5; V Tris-HCI, pH 7.5-9.5). Panel B: Standard reaction mixtures contained indicated concentrations of calcium chloride, in the absence (0) or presence (#{149}) of 25 M EDTA. Phospholipase A, activity was measured as described in Materials and Methods and is expressed as mol fatty acid released per mm per mg protein. Each value represents the mean ± SD of two experiments done in duplicate.
viability and percent acrosome-reacted the mean ± SD of three experiments.
are
reported
and
are
30
RESULTS Human sperm traction and cation
phospholipase exchange
2
A2, purified chromatography,
[‘4C]oleate-labeled, autoclaved E. co/i optimally alkaline pH (Fig. 1A). Figure lB shows that required for activity at pH 7.5. In the absence cium chloride mm/mg was
(open observed;
by acid exhydrolyzed
-J
at neutralcalcium was of added cal-
z
circles), a basal activity of 3.2 p.mol/ this was eliminated by the addition
0
4
I-
C
0
E
20
0
I1J
E
> 0
C >-
of 25 p.M EDTA tion of added EDTA (closed ner
with
chloride mM were
calcium circles)
up
stant between was further
to the
to
100
mixture.
chloride increased p.M
Activity
as a func-
in the presence in a dose-dependent
calcium
chloride,
of 25 p.M man-
remaining
100 p.M and 1 mM calcium chloride. increased by the addition of 2.5 mM
and
remained
calcium chloride; inhibitory.
The
reaction
in vitro
enzymic
at this
optimal
concentrations
level greater
up
than
to 20
lipase
A2 is inflammatory. phospholipids
>
10
U
K
0
0 0.0
0.2
0.4
20 mM PLA2(Mg)
activity
and
the
in vivo
edema-in-
ducing activity of human sperm phospholipase A2 as a function of protein concentration are compared in Figure 2. Increasing levels of human sperm phospholipase A2 caused an increase in the weight of the injected foot pad (edema), indicating that the highly purified human sperm phosphoof E. co/i
‘Ii
conActivity calcium
with
I-
w
A similar in vitro
was
increase observed
in hydrolysis over
the
same
FIG. 2. In vitro enzymic (A) and in vivo edema-inducing activity (#{149}) of human sperm phospholipase A2 as a function of protein. Standard reaction mixtures containing the indicated levels of protein were incubated at 37C for 2 mm. Phospholipase A2 activity was measured as described in Materials and Methods and is expressed as nmoles fatty acid released per mm. Each value represents the mean ± SD of two experiments done in duplicate. Enzyme-induced edema was measured by weighing the hind limbs of mice after iniection of human sperm phospholipase A,; edema is expressed as the mean ± SD (n = 6). Alkylated phospholipase A, (0.4 g) was tested for enzymic () and edema-inducing (0) activity.
ROLE
OF
HUMAN
SPERM
A2 IN FERTILIZATION
PHOSPHOLIPASE
hibited human a dose-dependent
100
dose
755
sperm phospholipase manner with an
of 50 mg/kg
PGBX
provided
A2-induced IC50 of
as determined
IC.)
of RET
50 >-
phospho!ipase A2 enhanced of 1 mM calcium chloride by change
(Fig.
SC),
in turbidity
respectively.
fusion, 78% of the tracer 5A). In contrast, fatty acid induced inhibited 0 0
PGB
sured same
10
5
(NM)
FIG. 3. Effects of prostaglandmn B1 (0) and PGB (#{149}) on in vitro phospholipase A2 activity. Standard reaction mixtures contained the indicated concentrations of the prostaglandin B, or PGB solubilized in DMSO. Phospholipase A2 activity was measured as described in Materials and Methods and is expressed as percent of enzyme control (100% = 16.5 p.mol/min/ mg). Each value represents the average ± SD of two experiments done in
fusion. Addition phospholipase by turbidity concentration
situ sperm pholipase
duplicate.
of PGBX enzyme
concentration
range.
Premncubation
of human
2, open
symbols). active
tegrity in vivo Most agents
or
results
indicate
is required
to produce currently
pase A2 activity, nonspecific; the cautiously, enzyme
These site
to alter
that
these
or
membrane
edema. used as inhibitors
obtained with the
a
bromide, are be interpreted
agents are used with crude [31]. PGBX, a base catalyzed
by the sodium
10 p.M concentration. A2 activity from washed “swim-up” procedure salt of PGBX (data
The effect of PGBX on membrane perturbations duced mouse administered
Similar sperm
of in vitro Figure 3 iland PGBX
effects on and sperm
[32] were not shown).
in vivo phospholipase was studied using
paw edema model. orally 60 mm prior
from vesicle that sperm that promotes
blocks is an
phospholipid phospholipase vesicle fusion,
expression effective
of enzymic inhibitor
on
sperm-oocyte
by 71%. A2 is a and that activity
of in vitro
phospholipase A2 activity, as well A2-induced vesicle fusion, we examined penetration,
an
The re-
and
and
in
as of phosthe effect
event
involving
a in-
(as an oil) on in vitro sperm phospholipase A2 activity. PGBX inhibits activity in a dose-dependent manner with an IC50 = 1.5 p.M; inhibition was near maximal with 5 p.M PGBX, whereas the monomeric prostaglandmn B, was only weakly at
of PGBX (5 p.M) to the fusion assay A2-induced vesicle fusion, as mea-
-J
0
oligomer of prostaglandmn B1, is a novel inhibitor and in vivo phospholipase A2 activity [19, 20]. lustrates the effects of prostaglandin B1 monomer
inhibitory phospholipase
enzyme-induced hydrolyzed (Fig. during calcium-
40
of phospholi-
including 4-bromophenacyl results of such studies must
especially when cell preparations
catalysis
cell
[1-’4C]-PE was was not released
SB) or efficiency
sperm
phospholipase A2 with the alkylating agent, 4-bromophenacyl bromide, resulted in almost complete inhibition of both in vitro enzymic and in vivo edema-inducing activities (Fig. functional
this
or RET, by 62% or 39%, respectively. of PGBX inhibited enzyme-induced
lease of [1-’4C]linoleate These results indicate membrane perturbant PGBX coordinately vesicle fusion. Because PGBX
(Fig.
During
vesicle fusion by 89 or 70%,
observed
A2-induced an enzyme-in-
Figure 4 shows that PGBX, to enzyme (0.4 p.g), in-
I-
z 0 C, IaJ
> 0
20
w
K
1L
0
0
10
PGBx
25
50
(mg/kg)
FIG. 4. Effect of PGB, on edema-inducing activity of human sperm phospholipase A2. The indicated doses of PGB. in 50 p.1 of DMSO were orally administered to the mice 60 mm prior to phospholipase A, injection (0.4 p.g) into the hind foot pad. Edema was measured as described in Materials and Methods and is expressed as the mean ± SD In = 6).
FRY
756
ET AL. TABLE
a
100
Li UI Li -J Li
a
3
1.
Effect
of PGB,
on sperm
penetration
of zona-free
hamster
oocytes.’ PGBX
Oocytes
(p.M)
exposed
0 10 25
50
I-
15 28 28 27
50
I-
Data
I.-
K
0
%Penetrate&’
No. penetrated
are the
mean
“5’ Penetrated
of three
oocytes
=
SD
±
93±22
14 14 3 1
50±
7
11 ± 4±
4 4
experiments.
penetrated
x 100
exposed
oocytes
0.5 0 0
as measured
a O
Cellsoft,
0.3
with
treatment. The effect by 0.0 100
K Li
flow
of
on
PGBX
was
acrosomal using
[27]
orescemnated untreated
pea lectin (Table sperm were viable,
FITC-PSA
in the
acrosome calcium
reaction. ionophore
cap
semen
NY),
region
an
affected
status
was
acrosomal
(CASA, by
this
monitored binding
2). Ninety-seven of which 29.9% to indicate
Sperm A23187,
analyzer
not
flu-
percent of did not bind
completion
of the
capacitated in the presence which is known to induce
of the
in vitro acrosome reaction [30], showed a 20% decrease in viable sperm compared to all other samples and resulted
50
(data little
0 PGB(zM) CaCI
Resources,
cytometry
in a 70%
E
a computer-assisted
CRYO
0
2
PLA2
0
0
5
10
-
+
+
+
+
-
-
+
+
+
acrosome induced
FIG. 5. Effect of human sperm phospholipase A2 ± PGB on phosphovesicle fusion and release of fatty acid from vesicle phospholipids. Phospholipid vesicles composed primarily of phosphatidylserine with trace amounts of (1-’4Cllmnoleate-labeled phosphatidylethanolammne were incubated with or without 10 p.g human sperm phospholipase A2 ± PGB, (1050 p.M) and 1 mM calcium chloride at room temperature for 10 mm. Fusion was monitored by the change in O.D., (panel B) or efficiency (E) of RET (panel C). Aliquots (0.5 ml) were removed and lipids were extracted to measure (1-’4Cllmnoleate release during fusion. In panel A, activity is expressed as percent fatty acid released (cpm free fatty acid/cpm total lipid x 100), Data are the mean ± SD of duplicate determinations. lipid
membrane shows that was inhibited
increase
perturbations and subsequent fusion. Table 1 hamster oocyte penetration by human sperm in a dose-dependent manner by PGB; 10 p.M
inhibited penetration by 50%. In previous experiments, the same concentration of PGBX (10 p.M) inhibited in vitro phospholipase A2 activity and vesicle fusion by 91% and 63%, respectively. No change in the inhibition by PGBX
of acrosome-reacted
not shown). to no effect
PGBX,
on sperm
reaction, the reaction
and
sperm
over
at all concentrations viability. PGBX did
control
tested, not block
at a 50 p.M concentration by approximately 40% over
had the
actually control.
Curiously 10 p.M PGBX did not significantly influence the percentage of viable sperm that were acrosome-reacted. Thus, the ability of PGBX to prevent penetration does not appear to be mediated by altered viability or acrosomal status. The results obtained by flow not shown) by means scribed
by Cross
cytometry were of fluorescence
also confirmed microscopy
(data as de-
et al. [30].
DISCUSSION The
present studies demonstrate that highly purified human sperm phospholipase A2 induces membrane perturbations such as vesicle fusion in vitro and edema in vivo. Expression of enzymic activity was required for these membrane perturbations as demonstrated by use of the covalent
PGBX
was
observed
and
washed
when once
sperm with
were fresh
pretreated medium,
with and
25 p.M PGBX
a second
wash-
ing resulted in only a moderate relief of inhibition; penetration increased from 11% to 50% of control values (data not shown). These results indicate that PGBX is not readily removed once it interacts with human sperm. In these experiments,
the
percentage
of motile
sperm
(75%
to 80%),
TABLE
2.
Effect
of PGB,
on human
sperm
PGB (p.M)
No. sperm evaluated”
% Viablec
0 10 50 Data
5180
±
183
96.9
±
5354
±
144
93.4
±
5312
±
94.2
±
are the
mean
141 of three
are
evaluated
for
bSperm
experiments binding
acrosome
reaction. % Viable and acrosomereactedd
3.0 2.5 2.5 ±
29.9
±
27.8
±
38.7
±
3.3 3.6 5.9
SD.
of fluorescent
molecules
by flow
cyto-
metry. Viability
dpercentage
is based on nuclear exclusion of propidium of viable sperm lacking FITC fluorescence
iodide. in acrosomal
region.
ROLE
inhibitor, kylates
Alkylation
coordinately
edema-inducing dicating brane
activity
that catalysis perturbations
reported
HUMAN
SPERM
PHOSPHOLIPASE
4-bromophenacyl bromide, which selectively active-site histidine of isolated phospholipases
the
A2 [33].
OF
inhibitor
inactivated
of the
sperm
al-
enzymic
and
phospholipase
is necessary to promote the memresulting in edema. Similarly, PGBX, a of in vitro
and
in situ
phospholipase
fuged found
A2
757
at 100 000 x g [3]. Membrane-associated to be equally distributed between
acrosomal
A2, in-
FERTILIZATION
A2 IN
and
contact
critical
tion, was
and also
inner
acrosomal
membranes-regions
to the
acrosome
reaction,
oocyte
sperm
phospholipase as determined
fertilization [3]. Human largely membrane-associated
differential
activity plasma/outer
the
centrifugation
of sonicates;
was of
penetra-
however
A2 by
the
distri-
activity sogenic mately
[19, 20], blocked enzymic, edema-inducing, and fuactivity of the human sperm enzyme to approxithe same extent. That PGBX also inhibited human
bution of the activity among subcellular membrane fractions was not reported [4]. During the ionophore A23187induced acrosome reaction, a 200% increase in mouse sperm
sperm
penetration
phospholipase
of zona-free
affecting viability or blocking that cellular phospholipase post-acrosomal tilization. The
hamster
acrosomal A2 activity
membrane
acid-extracted
reactivity, may have
suggests a role in
similar bilization
associated
with
lipids
interactions human
weight
sperm
phospholipase
of approximately
14000
by SDS-PAGE and gel permeation shown). Thus, the human sperm
and
membrane-perturbing
molecular-weight calcium
requirement
an
unusual
biphasic
response
Basal activity suppressed
(in the absence to zero by the
for
of calcium
sperm
catalysis,
to added
manner chloride
been
reported
for
studies
medulla
of snake
has
two
optima
venom
Recent
A2 gives response
a
crystallographic
reports
and
hamster
[16]
confirmed
sperm hamster
arachidonate some reaction, phospholipase
A23187
are
[39].
mobilized the sensitivity
A2 activity
Because
during of
in
to micromolar
fusion
sperm the
event.
on catalysis interactions.
provided
sperm.
conflicting
the
and
acadrenal
not
results.
bromide reaction
However,
port that 4-bromophenacyl human sperm acrosome
diated by calcium interaction with different binding sites to selectively influence catalysis. Increases in intracellular calcium have been measured during the human sperm acro-
ionophore
during
neurotransmitter release from Indeed, the fusogenic activity a neurotoxin that inhibits
of sperm with 4-bromophenacyl hibited the in vitro acrosome
penetration
calcium
the
activity
simply
have been used to probe the A2 in sperm-oocyte interactions,
have
inhibitory
follicular reaction
A2 among
acids and lysophospholipids of catecholamine from
dependent protein
Several inhibitors sperm phospholipase
ited
reaction induced by human the porcine sperm acrosome
was
in situ mophospho-
tro vesicle fusion and perturbs membranes in the mouse foot pad to induce edema. Alkylation of the enzyme with 4-bromophenacyl bromide coordinately blocked the enzymic and edema-inducing activities, suggesting that such
slightly
some during
A2 in this fatty release
and promote vesicles [42]. 3-bungarotoxmn,
[37], and it may be me-
domains observed
[3], which
in the sperm
of enzymic
of phospholipase
The cis-unsaturated cumulate during the
these
(100000)
A2 have
the presence of two calcium-binding is possible that the biphasic response
of phospholipase activation
perturbations are due to nonspecific
ob-
at 2.5to cal-
platelets [35] and C found in bo-
x-ray
phospholipase
con-
the other response
from bovine phospholipase [36].
1B).
were
noted
synaptic transmission, has been correlated with toxin-associated phospholipase A2 activity [43]. Our studies clearly show that human sperm phospholipase A2 stimulates in vi-
exhibits (Fig.
was
reaction in vitro are consistent with the involve-
medulla [40,41] brain synaptic of snake venom
which was (25 p.M), re-
a high-molecular-weight
phospholipase A2 isolated phosphatidylmnositol-specific adrenal
low-
but
phospholipases a similar biphasic
[22],
most
to micromolar
and
not to
enzyme
and
activity
4-fold increase from human
[6]. Distribution
ment
venoms, to morequire-
calcium
served: one between 50 and 500 p.M and 10.0 mM added calcium. While the typical cium by low-molecular-weight smooth hyperbolic curve
insect regard calcium
of added calcium), addition of EDTA
in a dose-dependent
centrations
A2 has
[34]. Like
A2, the
absolute
vine
A2 in vitro
to the reported of arachidonate
acrosome
as determined
and with
activity
phospholipases
an
has
fer-
chromatography (data enzyme is comparable
phospholipases A2 isolated from snake pancreas, and various human sources lecular size, stability to acid treatment,
sponded
without
membranes
a molecular
ment,
oocytes,
role
Treatment
or mepacrmne in guinea pig
more
of but
recent
in[15]
studies
re-
bromide had no effect on the reaction while mepacrmne was only
[18].
Para-bromophenacyl
of zona-free
[17], but had no effect sperm [44]. Mepacrine
bromide
hamster
oocytes
on oocyte is thought
inhibby human
penetration to inhibit
by phos-
fluid [38] induced
and by
pholipase A2 activity via a nonspecific interaction with the lipid bilayer to influence the enzyme-substrate interface [45]; 4-bromophenacyl bromide treatment inhibits a variety of
calcium
and
enzymes
both
A23 187-induced vitro human levels
acrosperm
of calcium
could provide a mechanism to regulate expression zymic activity during the acrosome reaction. Mouse sperm phospholipase A2 is predominantly ciated with subcellular membranes; more than 90% activity was sedimented when sperm sonicate was
of en-
dine, the due
other
discrepancies to nonspecific Recent studies
is a novel assoof the centri-
than
methionine,
cystemne
observed
with
effects demonstrate
inhibitor
interacts directly in a substrate-independent lated neutrophils and
PGBX
phospholipase
and/or
of
these
alkylating [31].
agents
phospholipase
the enzyme manner [19]. endothelial cells,
histi-
Therefore
are
probably
in cellular systems. that the PGB, oligomer,
in vitro with
A2 by residues
PGBX,
A2 activity. to inhibit activity In agonist-stimuPGBX suppressed
758
FRY
arachidonate
mobilization
duction,
measures
and
of in situ
Our results demonstrate sperm phospholipase whereas prostaglandmn centrations
(IC>
pholipase dependent pad
and
corresponding hydrolysis lytic
factor
phospholipase
A2 activity
50 p.M).
also
PGBX
blocked
[20].
sperm
and
vesicle
fusion.
its
inhibition
In our
by
studies,
were
PGBX
for human
fusion. Mammalian
sperm
phospholipase
fertilization
acrosomal
culminates and sperm
sperm-oocyte
a
A2 in membrane in the
fusion
12. Conway
of action
interactions.
in such
cellular
not
penetration
correlates
pholipase
A2 activity in vitro. A
phospholipase
with
A2 activity
the
effect
its inhibition dose
10-p.M
in vitro
of
by 96%
and
blocked
MN,
A2 in mammalian
tion.
CW,
Cytometry for
like
functional Core
performing
Daniel
Graff
We would
also
to thank studies.
Facility,
Ma.s.se
analysis
by flow
and
1985;
Center.
Cancer
Virginia
assistance
K.H. White
21.
against
pivotal
Reprod
Dcv
Commonwealth
University,
AC. Membrane and
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1973;
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JK. East J, Seyler
3. Thakkar
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Biochim
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MD,
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1989;
Polis
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Franson
4. ThakkarJK, with
human
Reprod
Franson
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1984;
Ada
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RC. Modulation
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12:167-177.
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PJ, Moani
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rosome 10. Fleming
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KO.
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by A23187.
of biological
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H, Cavt-ac JC, Pontonnier
A, Ribbes
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J.& The
8. Creutz
and
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and
Hoekstra
BJ, Van
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ac-
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phos-
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