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

Biophys

Acta

of fertilization.

1973;

Science

JK. East J, Seyler

3. Thakkar

spermatozoa.

Biochim

Rosenthal

MD,

acid

mobilization

Acts

1989;

Polis

BD,

Franson

4. ThakkarJK, with

human

Reprod

Franson

sperm

1984;

Ada

1983;

RC. Modulation

membranes

phospholipase

Kwong

by divalent

cations

and

A, activity calcium

12:167-177.

acrosome

reaction

membrane

of mammalian

model

spermatozoa.

of sperm Gamete

Res 1985;

Res

1974;

CanJ

Biochem

acyl ity

PJ, Moani

of human 7. Luv

elicited

fusion

aggregated 9. Meizel

by svnexin.J

S, Turner

rosome 10. Fleming

reaction, AD,

fertilizing

KO.

of phospholipases

by A23187.

of biological

CE. Cia-unsaturated

H, Cavt-ac JC, Pontonnier

A, Ribbes

for the activation

sperm

J.& The

8. Creutz

and

JP, Man.sat

L. Evidence

fatty Cell

Biochim

acids

Bio

capacity

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fusion

acrosome

E, Nelson

1987;

acids.

of various spermatozoa

35. 36.

919:255-265.

Physiol

MM.

of chromaulln

and

Hoekstra

BJ, Van

the hamster

FEBS

Lett

lipids

on the

with

1983; special

sperm

ac-

161:315-318.

acrosome

reference

to the

hymol-

Obst

Gynaec

LJD. CharacA,; evidence

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Mol

1989;

B, inhibit

in vitro

phos-

1006:272-277.

and

B, inhibit

endothelial

cells.

arachidonic

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Biophys

GL.

PGB,

An oligomeric properties.

derivative

Physiol

metabolism

of pros-

Chem

and

Phys-

by phagocytic

polymorphonuclear

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cell.

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for

total

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extraction

and

purification.

37:911-917.

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method

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the principle

D, Pagano

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the quantification

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H, Ueno

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Anal

Biochem

energy

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20:4093-4099.

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22. Franson

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Role of human sperm phospholipase A2 in fertilization: effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration.

Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined. Highly purified enz...
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