Effects of Phorbol Esters and a Diacyiglycerol on the ... - CiteSeerX

BIOLOGY

OF

36, 617-627

REPRODUCTION

Effects

of Phorbol Acrosome

MICHAEL

(1987)

Esters and a Diacyiglycerol

Reaction

A. LEE,

Induced

GREGORY

Department

by the Zona

S. KOPF,

of

University

and

Obstetrics

and

of Pennsylvania Philadelphia,

on the Mouse

Sperm

Pellucida1

BAYARD

T. STOREY2

Gynecology

School

Pennsylvania

of Medicine 19104

ABSTRACT Capacitated mouse sperm undergo the acrosome reaction when bound to isolated, mouse resembles, in part, ligand-receptor-mediated such processes have been shown the effects of phorbol esters, zona-induced acrosome reaction 13-acetate (TPA) chlortetracycline assay

with

cence on accelerated appearance

on

the

in part by protein kinase C-catalyzed activators of this kinase, on both At concentrations up to 10 pM,

examined.

the time course assay. Capacitated,

anterior

head;

fully

of

the spontaneous acrosome-intact

acrosome-reacted

nM

TPA

the fluorescence zona-induced are

mimicked

patterns, designated acrosome reaction, which by

60

nM

protein phosphorylation, the spontaneous and the 12-tetradecanoyl phorbol-

acrosome reaction as scored by the sperm display Pattern B in the CTC sperm

display

Pattern

AR

with

no fluores-

acrosome reaction was markedly of Pattern AR was retarded. The on the head and which marks an

between Pattern B and Pattern AR in the controls, was loss of Pattern B at early times post-binding to zonae. The to zonae was retarded by 65 nM TPA to the same extent

The transitions between define two stages of the of 65

were

in suspension and the zona-induced zona-induced acrosome reaction in the that occur in some somatic cells. Since

head. The time course of the loss of Pattern B in the zona-induced by 65 nM TPA as compared to controls, whereas the appearance of Pattern S, which is characterized by punctate fluorescence

intermediate state same extent as the times post-binding

effects

to be mediated which are potent

had no effect on (CTC) fluorescence

fluorescence the

spontaneous acrosome reaction intact zonae pellucidae. The exocytotic processes

accelerated by 65 nM TPA to the disappearance of Pattern S at later as the appearance of Pattern AR.

the B-to-S and the S-to-AR are affected in opposite

4-I’3-phorbol-12,13-didecanoate

(4-13-PDD)

transitions, directions by while

the

therefore TPA. The

4-a

isomer

is

without effect. Such stereospecificity is similar to that reported for the activation of protein kinase C. The diacyiglycerol, 1-oleyl-2-acetylglycerol, which is also known to activate protein kinase C, mimicked the effects of TPA and 4-13-PDD on the time courses of the B-to-S and S-to-AR transitions. These results suggest that protein kinase C may play an intermediary role in the zona-induced mouse sperm acrosome reaction.

INTRODUCTION

Capacitated reaction at and zona

at

mouse sperm undergo a slow rate spontaneously

a much pellucida

more rapid rate when of the egg (F!orman

1982a). The acrosome reaction the zona is induced by the zona by the compound 3-quinuclidiny! with unique potency (Florman 1982b). The acrosome reaction

the acrosome in suspension bound and

to the Storey,

of sperm bound to itself and is inhibited benzi!ate (QNB) and Storey, 1981, inducing activity

of the mouse zona pellucida

zona has been shown to reside with the g!ycoprotein ZP3 (Bleil and Wassarman,

1983), the sperm-binding 1983, 1986;

same glycoprotein that also possesses activity (Bleil and Wassarman, 1980, Florman et a!., 1984, Florman and

Wassarman, biological

1985;Wassarman activities have

eta!., been

1985). These differentiated

two ex-

perimentally. Binding to zonae was shown not to be affected by QNB, whereas induction of the acrosome reaction was inhibited (Florman and Storey, 1982a). The sperm-binding a discrete size class 3400-4500, molecule

Accepted September 29, 1986. Received July 31, 1986. ‘This research was supported by NIH grants HD-06274 and HD19096. 2 Reprint requests: Dr. Bayard T. Storey, Department of Obstetrics and Gynecology, Medical Labs 339, University of Pennsylvania, Philadelphia, PA 19104-6080.

Wassarman, ZP3, on

617

which with

the

of acrosome a!., 1984),

activity of 0-linked

retention 1985). other

can

has been localized o!igosaccharides

be

cleaved

of

activity

from

Minimal proteolytic hand, resulted in the

reaction-inducing implying that this

the

to of Mr parent

(Florman cleavage complete

activity (Florman activity is conferred

and of loss et by

LEE

618 functional groups on the and/or a very strict steric charide

groups

chain. The binding have requirements. receptor trypsin

protein backbone of ZP3 configuration of oligosac-

imposed

by

the

sperm-associated also been shown There appear to

sites: (Saling,

intact

polypeptide

receptors for zona to have distinct steric be two classes of such

one class is sensitive to inhibitors 1981) and the other is sensitive

using

125

I-labeled

sarman, 1986), in agreement of evidence that mouse mouse

zona

(Sa!ing

and

with

with sperm

a fully

Storey,

ZP3 the bind

intact

1979;

Saling

(Bleil

plasma et al.,

and

Was-

extensive to the

body intact

membrane

1979;

Florman

is an important

cells Ca2

is -

class.

apparently exocytotic

plays processes

protein phosphorylation and phospholipid-dependent

designated zuka et al.,

that

a!.,

1986).

diacy!g!ycerol

protein 1984; This

a key role in in somatic

catalyzed protein

by the kinase,

kinase C (Nishizuka, 1984; NishiBlumberg et a!., 1985; Kimura et enzyme

generated

is activated by

either

ligand-induced,

by

1,2-

receptor-

mediated polyphosphoinositide hydrolysis (Lapetina and Miche!!, 1973; Berridge and Irvine, 1984; Cockcroft, 1984; Sekar and Hokin, 1986; Smith et a!., 1986), latter, potent

or by a variety of the phorbol esters and useful (Nishizuka,

of

exogenous agents. Of the have proved particularly 1984; Blumberg et al.,

sequential

stages

by

the

as a fluorescent

probe

time first

courses of these stage and retard

two the

stages: second

AND

METHODS

Reagents

Glutaraldehyde solution from

was obtained Po!ysciences, Inc.

12-Tetradecanoy!phorbo!-1 racyc!ine (CTC), bovine

as a 25% (Warminster,

Phorbol-12,

13-didecanoate

1 3-didecanoate (OAG)

Jolla,

CA).

(4-a-PDD),

P.

F.

Nutley,

NJ.

4-13-phorbo!1-oleyl-2-acety!-

purchased

from

Calbiochern

for

Sorter,

from 4-a-

and

culture

from Flow Laboratories nuclidiny! benzilate (QNB) Dr.

were obtained Louis, MO).

(4-j3-PDD), were

Water

aqueous PA).

3-acetate (TPA), chlortetserum albumin (BSA), and

hyaluronidase (bovine, testicular) Sigma Chemical Company (St.

media

(McLean, was the

was

(La

obtained

VA). generous

Hoffmann-LaRoche

3-Quigift of

Laboratories,

9-Amino-3-chloro-7-methoxyacridine

(ACMA) was generously provided in purified Prof. C. P. Lee, Department of Biochemistry, State University School other reagents, obtained of analytical grade.

of Medicine, from various

form by Wayne

Detroit, suppliers,

MI.

All were

Media The culture medium (CM) used in all experiments was a modified Krebs-Ringer bicarbonate solution described by Toyoda et al. (1971), containing 25 mM sodium glucose, prepared

lactate, and 20 without

1 mM mg/rn! BSA,

a 0.20-pm in 20-rn!

filter aliquots

(Nalgene) for single

as needed, through a CO2 /95%

(1985).

two

the the

MATERIALS

has

into

(CTC)

1985). In this paper we report that, to zonae, phorbol esters are potent

stage.

were and

resolved

Storey, bound

perturbants of they accelerate

1985; Brock et al., 1985; Sharkey and Blumberg, 1986). If the zona-induced acrosome reaction were mediated by this mode of intracellular signaling, then one would expect perturbation of this process by phorbol esters. The zona-induced acrosome reaction been

ch!ortetracycline

(Lee and in sperm

glycerol

action of hormones with plasma membrane surface receptors on somatic cells. Sperm-zona pellucida interaction may, therefore, represent a process analogous to ligand-receptor interactions characterized in many somatic cells, of which ligand-induced One reaction receptor-mediated

use

12,

and Storey, 1982a; Storey et a!., 1984; Saling and Lakoski, 1985; Lee and Storey, 1985). The apparent specificity of sperm-zona interaction parallels the strict specificity known to be required for the inter-

exocytosis

AL.

of to

inhibitors of galactosy! transferase (Shur and Ha!!, 1982; Lopez et al., 1985). QNB does not affect sperm binding, as pointed out above, nor does it perturb the site sensitive to trypsin inhibitors (Benau and Storey, 1986). The sites for zona binding have been shown to be on the sperm plasma membrane by direct autoradiography

ET

sodium pyruvate, 5.57 mM BSA. Medium CM was first sterilized by passage through and use.

supplemented 0.20-pm filter,

then frozen at -80#{176} C Aliquots were thawed

with and

BSA, gassed

resterilized with 5%

air to pH 7.4.

Gametes Procedures based were All

for on those described handling

the

preparation of Inoue previously of

gametes

of and by

mouse

gametes

Wolf (1974, Lee and.

was

done

1975) Storey at

37#{176}C

PHORBOL

under

silicone

oil

Corp.,

Midland,

MI)

CO2 /95% were obtained (Harlan

Eggs

for

mature (Harlan

Sprague

zonae

had

with

Inc.,

were

Sperm

of

10.0

IU

and Zonae

(hCG;

mice super-

containing the 10-fold dilution

IN).

at

Binding

were prepared a narrow-bore

diameter

of

were washed adherent egg

the

to

Pellucidae

by forcing micropipette

zona-intact

3 times cytoplasm

egg).

comulus-free (i.d. The

isolated

eggs 0.75

=

zonae

in medium CM to remove and resuspended in 100-200

for

at 60

a concentration mm in the above

the appropriate 1 mM OAG;

60

compound nM 4-a

were added formamide

from (DMF)

0.1%

final

in the

of 10-25 X 10 medium containing

DMF

in dimethylwas present

alone

the control determinations. Comparative experiments showed that this concentration had no effect on the ability of the sperm

was

added

at in

control of DMF to bind to

was adjusted to 2 X 10 of the sperm suspension

cel!s/m! to a

100-pl drop containing 100-150 mechanically isolated zonae pel!ucidae plus the same compound at the concentration present during capacitation so as to maintain a constant concentration. The point of addition be the

of sperm zero time

compound so that the

sperm with the compound was at of the sperm-zona binding step. the test compound was present but was omitted from the drop

zonae, of the

allowed time

to the isolated zonae point in the experiments

was

sociated pM QNB mm

resulting compound

to

the

bind

zonae

taken to described

sperm. In was added

post-binding

in

an approximately as the sperm were

to the with

zonae

for

bound

a second set of to the sperm-zona

of sperm

Fluorescence

15

sperm

mm, were

experiments, suspension

50 60

to zonae.

Assays

The binding lucidae was (400 X) using

of sperm to the isolated scored by phase-contrast a Nikon Optiphot microscope

interchangeable optics. The

phase contrast bound sperm were

zonae pe!microscopy equipped

and then

epifluoresexamined

by epifluorescence microscopy (400 x) for patterns of CTC fluorescence that reflect the state of capacitation and the stages of the acrosome reaction, according to the method of Ward and Storey (1984). At the designated time point, 5 p! of a 500-pM CTC solution aded

washed

structurally intact zonae and undergo the acrosome reaction. Incubation of sperm with these test compounds during capacitation was used in the majority of the experiments. After capacitation, the final sperm concentration by adding an aliquot

which

was

to be tested (65 nM TPA; or 4-13-PDD). The compounds

stock solutions such that DMF

suspension;

were

with cence

pl CM (Lee and Storey, 1985). Sperm suspensions were prepared from excised caudae epididymides as described by Lee and Storey (1985). Sperm were capacitated, cells/rn!,

test step,

washed 3 times in CM containing 50 pM QNB using a wide bore micropipette to remove any loosely as-

All incubations were carried out in medium CM containing 20 mg/m! or 4 mg/mI BSA under silicone oil at 37#{176}Cin a humidified atmosphere of 5% CO2 in air. Mechanically isolated, structurally intact zonae pel!ucidae through

the

added to the zonae. In one set of experiments, QNB was added to a final concentration of 50 pM in the zonae suspension prior to the addition of sperm. Sperm

Chemical

619

In some experiments, from the capacitation

virgin,

pregnant

Sigma

REACTION

first contact of the the zero time point In other experiments, during capacitation

(PMSG) (Gesty!; Disoynth, 36-56 h later by 10.0 IU

gonadotropin MO).

Intact

with

Indianapolic, from

ACROSOME

in the figures. was omitted

Corning

Swiss Webster Mice were

Inc.).

THE

Spermatozoa Swiss Webster

obtained

injections

Capacitation

Structurally

Dow

AND

equilibrated

Dawley

mare’s serum gonadotropin Chicago, IL), followed human chorionic Co., St. Louis,

been humidity. breeder

12-week-old) Dawley, i.p.

200,

100% retired

isolation

(10to Sprague

ovulated

Corning

that

air at from

5%

mice

(Dow

ESTERS

to

an equal

zonae

with

volume

(37#{176} C) slide. Glutaraldehyde pH 7.8) was immediately tration placed zonae.

of over The

100% patterns.

humidity This

of CM containing

bound

sperm

on

5-10

a pre-warmed

(12.5% in 1 M Tris HC1, added to a final concen-

0.1%. A covers!ip was then carefully the 10-p1 drop containing sperm bound to slides were kept in light-tight chambers at until mode

assayed of fixation

for CTC maintains

fluorescent the patterns

without change for up to 12 h. In parallel experiments, the uptake of the dye ACMA was examined in sperm bound to zonae for different time intervals. A 500pM stock solution of ACMA in DMF was prepared and kept in a light-shielded container. At the time of assay, 9.85 pl of CM containing 5-10 washed isolated zonae with bound sperm was added to a warmed slide. A 0.1 pl aliquot of the ACMA stock solution was added with stirring to give a final concentration of 5 pM ACMA. Glutaraldehyde (12.5% in 1 M Tris-HC1, pH 7.8) was then added to a final con-

LEE

620 centration the drop container

of 0.05%. A covers!ip was then placed over and the slides were held in a light-tight at 100% humidity, as described for the CTC

procedure. from 15

mm

Determinations of to 5 h post-fixation

ACMA fluorescence showed that the

fluorescence patterns were stable to at least 5 h. The slides were examined under phase contrast (400 X) and then under epifluorescence (400 X) for fluorescence patterns of CTC and ACMA on the zonae-bound sperm.

Details

of

emission

system

Lee

Storey

and

time with

epifluorescence

both

(1985).

course 2-3

the

for

and

have

been

described

For

each

time

point

determinations, sperm/zona

excitation

dyes

a

were

total

of

by in the

30

zonae

scored.

of

In

TPA

a previous

acrosome

Sperm

CTC

study

of

reaction

sperm

bound

zonae pellucidae, in two stages, and

on

calibrated

Storey, 1985).

to

induced isolated,

in

ultrastructural

course

of

capacitated

the

mouse

intact

studies

mouse occurred probes

(Saling

1979; Lee and acrosome-intact

a pattern of fluorescence anterior head and midpiece

and

Storey, sperm

characterized by separated by a dark

band over the postequatorial segment. This banded pattern was designated Pattern “B.” It disappeared with time of incubation of sperm bound to zonae and was replaced by a punctate, or spotty, pattern of fluorescence

over

With

time

further

the

head,

designated

of incubation,

Pattern

Pattern

“S.”

and for convenience have been designated of CTC fluorescence patterns. The first stage B-to-S transition. The second stage is the

S-to-AR transition. stages was examined.

The

effect

When sperm were incubated (Nishizuka, 1984) during both zona-binding sequences, both S-to-AR course bound started

for

the

of TPA

cell

population

1). This characteristic lag period, with structurally intact zonae 1982a; Lee and Storey, 1985, TPA, population

and

by

the was

loss of complete

more rapid loss of Pattern particularly at the earlier mirrored by a more rapid Pattern S, when compared

Pattern in 150

mm

(Fig.

consistently and Storey, was abolished

B from the sperm mm (Fig. 1). The

B in the presence of TPA, times of incubation, was rise in the percentage of to the control (Fig. 2).

The presence of TPA therefore transition. No effect of TPA zonae was observed during these

the

in 180 observed (Florman 1986),

accelerates on sperm experiments.

the B-to-S binding to

effect was seen with TPA on the which marks the second stage of acrosome reaction. The percentage

displaying Pattern S remained elevated later incubation times when compared to

control,

and

did

not

decrease

below

30%

(Fig.

2).

The percentage of sperm displaying Pattern S in the presence of TPA remained constant for up to 260 mm, as determined in a separate set of incubatmons for extended times. Pattern AR, which approached 90% zero

as the in the

only 70% mained at

percentage control

of Pattern experiments

in the presence this percentage

S dropped towards at 210 mm, reached

of TPA (Fig. for up to 260

3) and reWhen

mm.

100

80

S disappeared

and was replaced by Pattern AR, corresponding to the completed acrosome reaction and characterized by dark heads and bright midpieces. The two stages of the zona-induced acrosome reaction occurred in sequence, in terms is the

completion

of sperm during the

Patterns

that the reaction with fluorescent

1979; Saling et a!., With CTC, capacitated

showed bright

time

structurally

we found as determined by

Fluorescence

the

AL.

The opposite S-to-AR transition, the zona-induced

RESULTS Effects

ET

on

both

60

40

20

these 0

with 65 nM TPA the capacitation and the B-to-S and the

transitions were affected with regard to time (Figs. 1-3). The loss of Pattern B from the sperm in the unperturbed control system only after a lag period of 30 mm and reached

0

30

60

90120150180210240270

Minutes FIG. 1. The effect of 65 nM TPA on the time course of the loss of CTC fluorescence Pattern B in mouse sperm bound to isolated, intact zonae pellucidae. For each time point, a total of 30 zonae with 2-3 sperm/zonae were scored. Error bars Control points in the absence of TPA: presence of 65 nM TPA: (0).

represent standard deviations. Experimental points in the

(.).

PI-IORBOL

ESTERS

AND

100

THE

ACROSOME

REACTION

strated that mechanically isolated zonae, incubated with phorbol esters for at least 1 h, still retain their ability to induce the acrosome reaction of bound sperm with a time course similar to that seen with control, untreated zonae (Endo et al., 1987b). In experiments where 65 nM TPA was absent

80

60

%s

during

capacitation

added TPA

40

0

I#{149}

0

30

I

11

60

1#{149}

90

I’I#{149}

I#{149}

sperm

I

120150180210240270

Minutes

reaction

in separate

was

experiments,

increase

there

in the

extended at the

monitored was

percentage

times, TPA

after no

further

of AR

there was concentration

300

mm

signifi-

patterns.

no effect used

Even on sperm in these

experiments. Pattern AR was observed in the sperm at earlier times in the presence of TPA when compared to the control sperm, which is the result expected from

an

states

characterized

elevation

(Fig.

2).

The

in the by increase

time was slower absence (Fig. 3).

percentage

Pattern in

in the

percent

presence

of

which (Wolf,

is optima! for capacitation 1979). Since BSA has been and

earlier

pattern

experiments out in medium

(Dicker

not

due

TPA

than

on

the

zonae.

same

as those

effects

were

of

CTC

the controls were

no

longer

fluorescent

in the carried

absence out to

incubation with TPA did its effect could be reversed

its effect

occurred

which

occurs

only

in the

was further exon the spontanein mouse

in medium CM in the absence at a rate far slower than that induced (Florman and Storey, 1982a). The

sperm

of zona by zona spontane-

reaction was unaffected by TPA at as high as 10 pM. No changes in the of the CTC fluorescent patterns

It has

experiments

been

80

were BSA,

and fertilization reported to bind

1980),

100

in its

to those observed with 20 mg/ml BSA (Figs. Both mediation and perturbation of the TPA by BSA can therefore be ruled out. effects of TPA on these transitions were due effects of this phorbol ester on the sperm, and to effects

TPA

This last point the effect of TPA

reaction,

ous acrosome concentrations rate or quality

with

were carried out to assess whether the differential effects of TPA on the B-to-S transitions could be due to the time-dependent binding of the phorbo! ester by BSA. When the BSA concentration was reduced to 4 mg/rn!, the effects of 65 nM TPA were essentially identical 1-3). effects The to the

the

effects of of CTC

times

AR

shown in Figures 1-3 CM containing 20 mg/rn!

Rozengurt,

by

acrosome

suspended protein protein

were

courses

that

zonae. testing

similar courses

were

of intermediate

S at these

The carried

TPA

and of

sperm

sperm in Figures 1-3. If during capacitation but was 10-fold upon addition of

that prolonged the sperm, that

dilution,

ous

the time

when

time point), The time

were the same as for These experiments

demonstrate not damage presence amined

added

changes

zonae, the

patterns of TPA.

by

acrosome

to

observed:

FIG. 2. The effect of 65 nM TPA on the time course of the appearance of CTC fluorescence Pattern S in mouse sperm bound to isolated, intact zonae pellucidae. The conditions were those for Figure 1. Absence of TPA (U); 65 nM TPA (0).

at these motility

pattern

was

shown for the TPA-treated 65 nM TPA was present diluted approximately

20

cant

but

to zonae (zero were expressed.

fluorescent

the

621

demon-

60

%R 40

20

0 0

30

60

90

120150180210240270

Minutes FIG. 3. The effect of 65 nM TPA on the time course pearance of CTC fluorescence pattern AR in mouse sperm isolated, intact zonae pellucidae. The conditions were those 1. Absence of TPA (a); 65 nM TPA (0).

of the apbound to for Figure

LEE

622 were

observed

at

more than two needed to affect bound

to

this

zonae.

When

motility pM TPA

and morphology were unaffected

mm. These

results

interaction the

that

proteins

occur

and

the

B-to-S

the This

is a potent acrosome

transition,

is

sperm with 10 to 300

up

post-binding and

in order

S-to-AR

leading to were blocked.

with QNB, which the zona-induced

controls,

a specific,

zona

must

B-to-S

to

the for

sperm TPA

transitions.

implication were valid, it would effects of TPA would be blocked interactions reaction

which than that of sperm

in groups treated for time periods

the

membrane

affect

concentration,

compared

imply

between

plasma

TPA

orders of magnitude greater the CTC pattern transitions

If

to this

be expected that the when the sperm-zona zona-induced supposition

acrosome was tested

and specific inhibitor of reaction at its first stage,

but

does

not

affect

sufficient all of the

in the sperm

acrosome reaction by (Figs. 1-3). It has been must be present between

absence population

of

QNB to complete

have the

Pattern that 20 min

AR QNB after

progressing to shown previously zero time and

acrosome ionophore

reaction concerns Ca2 A23 187 at 5 pM

accelerate reaction

The diva!ent cation has been shown to

markedly the zona-induced in sperm bound to isolated intact

acrosome zonae (Lee

and Storey, 1985). The two stages, B-to-S and Sto-AR, can no longer be resolved: the entire population of bound sperm undergo a B-to-AR transition within the time resolution of the CTC assay. This suggested that A2 3187 might overcome the retardation of the S-to-AR transition induced by TPA. When this the

was tested, same result

it was found in the presence

absence: only a B-to-AR The final percentage playing the AR pattern A23

187

of TPA

of

that 5 pM A23 187 gave of 65 nM TPA as in its

transition could be observed. the sperm population diswas 100% in the presence of

plus

TPA,

compared

alone

(Fig.

3).

to

70%

in the

presence

sperm

binding to the zonae (Florman and Storey, 1982ab; Storey et al., 1984; Lee and Storey, 1985). As shown in Figure 4, QNB is as effective an inhibitor of the B-to-S transition in the presence of 65 nM TPA as in its absence. The sperm bound to zonae retain Pattern B over the entire 210-mm observation period, a time nearly

ET AL.

Stereospecificity

The was

of

isomeric

examined

the

Phorbol

specificity using

Ester

of the

available

Effects

phorbo!

phorbol-12-1

ester

effect

3-didecano-

ate stereoisomers. TPA has the (3 configuration at the 4-hydroxyl group with trans fusion of the A and B rings. The corresponding stereoisomer, 4j3-phorbol-12,13-didecanoate (4-j3-PDD; 60 nM) had very similar AR patterns didecanoate other hand,

effects on the time courses of B, 5, and as 65 nM TPA. The 4-a-phorbo!-12,13stereoisomer (4-a-PDD; 60 nM), on the had no effect on the time-course curves

sperm binding to zonae for effective inhibition of the zona-induced acrosome reaction to occur. Addition of QNB 60 mm after sperm binding has no inhibitory effect, and the sperm to have been occluded

binding at this

site for QNB time by zona

(Florman et al., 1982). The implication sperm-zona interaction is complete at 60 binding. This would lead to the expectation added at this of TPA. This ment. resulted

Addition of in time-course

patterns those served

in

the

QNB

presence

is

closely

effect effect coupled

glycoprotein acrosome

of

not affect borne out

at curves

60 mm post-binding for the three CTC

65

nM

TPA

the action by experi-

identical

needed reaction.

additional pellucida

on TPA. TPA on

These results indicate the B-to-S transition is

interaction for

successful

aspect of and sperm

to also direct

of sperm induction

the interaction leading to the

and

zona of

-

80

%B

would was

of

to the

that postQNB

mm that

shown in Figures 1-3. This experiment as a control to show that QNB had no

antagonistic that the

An zona

time point supposition

100

appears protein

I

I

-

60 40

-

20

00

30

60

90

120150180210240270

Minutes

the

between induced

FIG. 4. Effect of 50 MM QNB on the loss of CTC fluorescence Pattern B in mouse sperm bound to isolated, intact zonae pellucidae. The conditions were those for Figure 1. Absence of TPA (#{149}); 65 nM TPA (0).

PHORBOL

of of

the CTC time-course

fluorescence curves

disappearance essentially

of the superimposable

1-3,

4-a-PDD

with

1984;

the

control

the TPA curves. The of the zona-induced sperm has the same

Blumberg

a!.,

activation stereospecificity

likely

this

THE

ACROSOME

1985).

Since

100

were Figures

80

curves

and

of protein (Niedel

enzyme

is the

target

kinase et a!.,

40

20

phorbol

C also 1983),

of these

disit is

agents

0 0

in

30

60

Effects

of

The kinase

physiological C is considered

intracellular to be the

from

hydrolysis phorbo! protein

100

1, 2-Diacyiglycerol

generated

membrane

regulator diacylglycero!

of protein moiety

65

nM

1985; courses

acrosome disappearance than with

If the sperm that

et a!., 1984; Brock et al., 1985). OAG, the concentration expected

Effect Associated

of

180

150

180

210

240

TPA

(Blumberg

Sharkey of the

and two

%S 40

et a!.,

Blumberg, stages of

1985;

20

Brock

et

1986), on the zona-induced

the

0

TPA with

on

Sperm the

B-to-S

was observed on of zonae pellucidae. ACMA

Fluorescent

sperm

100

a pKa within

120

1

210

240

-

0/

40

20-

0

.

0

is a weak base with 1983), accumulates

90

60-

Transition

1985). of 8.8

I’E’

60

-

80

Patterns

ACMA has been shown to for the first stage of the reaction (Lee and Storey,

30

Minutes

in

The fluorescent probe be an additional marker zona-induced acrosome This probe, which (Huang et al.,

11II1

0

reaction is shown in Figure 5. The rate of of Pattern B is somewhat more rapid TPA, but otherwise the effects of OAG,

1 mM OAG in the absence

-

60

The to

TPA, and 4-13-PDD are very similar. The B-to-S transition is accelerated while the S-to-AR transition is retarded. Incubations extended to 260 mm in the presence of OAG also displayed a plateau of both Pattern AR (7 0%) and Pattern 5 (3 0%) of the sperm population. As observed with TPA and 4-(3-PDD, no effect of suspension

150

polyphosphoinositide

(Nishizuka, 1984; Berridge, 1984). esters were acting by stimulating kinase C activity, it would be expected

(Nishizuka of 1 mM

mimic

120

-

80

an exogenous 1,2,-diacy!g!ycerol would have effects similar to TPA and 4-13-PDD. A diacy!g!ycero! frequently used as a test compound for comparison is OAG effect

90

Minutes

sperm.

mouse

a!., time

623

60

perturbation acrosome stereoisomer

the

REACTION

sets and/or

esters as do the various effects in other cell types (Nishizuka,

et

ester-induced plays such

AND

two

and AR patterns the curves of

on

for phorbol by these agents

that

5,

mimicking

4-#{237}3-PDDmimicking ot the two stages reaction in mouse specificity induced

patterns. The the appearance

for B,

ESTERS

IIIIIII

30

I

60

90

120

150

180

210

240

Minutes FIG. 5. Effect of 1 mM OAG on the time course of the loss of fluorescence B (top panel), the appearance of Pattern S (middle panel), and the appearance of Pattern AR (bottom panel) in mouse sperm bound to isolated, intact zonae pellucidae. The conditions were those for Figure 1. CTC

LEE

624 100

ET AL.

Pattern

5, was

reaction, through

80

60

obligatory.

defined by intermediate

B-to-S-to-AR.

This

a sequential

first-order

Completion Pattern state

AR, 5,

formulation

of the acrosome could with

is the

chemical

only proceed the sequence same

as that

reaction

written

B

As

0

30

60

90

120

150

180

210

Minutes FIG.

6.

Effect

of

65

nM

TPA

ACMA fluorescence Pattern intact zonae. The conditions (#{149}); 65 nM TPA (a).

on

the

time

A in mouse are those for

course

sperm Figure

of

the

bound to 1. Absence

loss

of

isolated, of TPA

the

reaction

mouse

fluorescence some. ACMA

because

This has fluorescence

time

sperm

course

of

of

been the

with

pH

within

a lower

designated was shown loss

heads

of

Pattern to follow

CTC

Pattern

enhanced the

acro-

distribution different with this

B both of 50

tion

that

both

(Lee

and

Storey,

support

probes

monitor

for the

the

earlier B-to-S

contentransition

1985). DISCUSSION

The acrosome the time patterns population and that

division reaction

of

the zona-induced mouse sperm into stages was originally based on

course of the changes in CTC fluorescence observed as the reaction proceeded in a of sperm bound to isolated zonae (Lee

Storey, 1985). an intermediate

The essence of such state, experimentally

a model defined

was by

B

of zona-bound sperm displaying the fluorescent patterns was fully consistent formulation, if the rate constants k1 and k2

model consisting regulated

for

But in the original study, means of distinguishing

there was between

and

consisting was seen

inhibitory

the first formulated decrease

k2.

of a single experimentally

effects

view.

cence patterns Pattern S at rapid decrease in Pattern intermediate that Pattern

no a

the zona-induced acrosome reaction of two sequential stages, with each stage by different regulatory processes, and a

as two sequential membrane changes. The data Figures 1-3 support the idea that the biologically active phorbo! esters and OAG have both stimulatory

1 for loss of CTC Pattern presence of TPA. Addition

further

of

B in sperm

for Pattern B. The coincidence of the time-dependent loss of Patterns A and B in both the absence and presence of TPA, and the inhibition of both losses by provide

concentration

model progress

pM QNB at zero time of binding completely inhibited the loss of ACMA fluorescence; the results with Pattern A were the same as those shown in Figure 4

QNB,

the

A. Loss of closely the

bound to zonae and to be associated with loss of the H+ permeability barrier across the plasma membrane (Lee and Storey, 1985). The effect of 65 nM TPA on the time course of ACMA fluorescence loss is shown in Figure 6. The time course is essentially identical to that shown in Figure in the absence and

proceeds,

falls monotonical!y with time; the concentration of S rises as it is formed from B, then falls as it is converted to AR; the concentration of AR rises after a lag time, then levels off as the reaction is completed. The

were similar. experimental acrosome-intact

AR

k2

k1

0

as:

F-

40

20

for

In above, The

regulated reaction whose by CTC fluorescence

on these terms these

effect

of

transitions

the agents

on the

sequential increase

distribution

and

in

support reaction k1 and

of fluores-

is observed as a more rapid increase in earlier times, with a concomitant more in Pattern B. This more rapid increase S at earlier times provides more of this state leading to Pattern AR. The result is AR is observed with TPA at earlier times

than in the control, although its rate of formation from the intermediate, as shown by the slope of the curves tracing the increase of AR with time in Figure 3, is less. The B-to-S transition is closely correlated in time course with the loss of ACMA fluorescence both in the presence and absence of TPA. Since the loss of ACMA fluorescence represents a loss of the permeability barrier of the plasma membrane, such that a pH gradient can no longer be maintained, the B-to-S transition can be defined operationally in terms of an “intact-to-leaky” transition for the plasma membrane over the sperm head. The next stage, S-to-AR, can

PHORBOL

then Since

be defined each of

operationally these transitions

ESTERS

AND

as “leaky-to-reacted.” comprises a group

THE

of

closely linked reactions, at least two plausible sites of regulation of the zona-induced acrosome reaction by phorbol

esters

may

kinase C activity in mouse sperm, these compounds

the

acrosome

reaction

processes reported,

do involve moreover,

boar

sperm

directed protein

be

postulated.

protein

has not yet been demonstrated potency and stereospecificity in affecting the zona-induced suggest

that

this enzyme. that this

(Adeyemo

at the kinase

Although

and

these

It has enzyme Koide,

identification C in mouse

regulatory

Studies

and characterization sperm are currently

of in

625

REACTION

compelling pe!!ucida Lakoski,

that mouse sperm bind to the with an intact plasma membrane (Saling 1985; Lee and Storey, 1985; Bleil

Wassarman, microscopy, zona

of

been recently is present in 1986).

ACROSOME

1986). When all sperm bound

of

whereas vitelline 1979).

zona-intact

examined to the

eggs

had

by outside

intact

those sperm that had penetrated the penspace were acrosome reacted (Saling et a!., The two CTC patterns corresponding to these

states of the mouse sperm were the B and AR patterns, respectively. At present, there is no firm evidence that, in the absence of perturbants, sperm displaying CTC Pattern the zona. It

S still remain bound to the outside of has been shown, however, that mouse

sperm

displaying

The effects interaction of

outside treated to the

of the zonae of mouse eggs that with TPA, a condition in which the full acrosome reaction does not occur

membrane,

since

the

on mouse pellucida effects

sperm require the and sperm plasma

are

manifest

at

60

nM

Pattern

TPA in the presence of zonae but are not observed at concentrations as high as 10pM in sperm in suspension in the absence of zonae. The presence of zonae

a!., 1987b). This implies ligand groups may mediate

pel!ucidae

division described

of labor above,

cellular for the

messenger zona with

is

also

necessary

for

the

OAG

effects

shown in Figure 5. These data support the concept that the sites regulated by phorbo! esters and diacylglycerol are affected only when the sperm interact with

a

physiologically

relevant

pellucida. Since ZP3 both sperm-receptor activities

(Bleil

be anticipated protein and required phorbo! remains

and

Wassarman,

that the the mouse

for the esters to be

specificity of that coupling

1980,

interaction sperm

this of

interaction the zona

receptor Both senger

which in

two types an intracellular

occupancy interact regulates

zona

1983),

with

to

of

The

zona is

concept receptor

ester-sensitive sites of a series of defined model based experimental

on this data, is

messenger molecules signal chain linking

protein

protein enzyme

the

kinase activity

kinase

C activation.

C, but involved

one in

mesthe

B-to-S transition, while the other messenger regulates enzyme activity involved in the S-to-AR transition. Definition of each of these transitions, or stages, of the still

zona-induced at very coarse

acrosome resolution.

reaction is, of necessity, The evidence is by now

another

reaction cellular

it would

between this plasma membrane

supports ligand/sperm

messengers. The simplest and consistent with the

one in participate

the

expression of the observed effects of and diacylglycerol. However, this demonstrated experimentally. The

interaction with the phorbol occurs through the generation cellular concept,

ligand,

is the zona protein that possesses and acrosome-reaction-inducing

electron of the

acrosomes,

progress. of TPA the zona

zona and and

may

mediate

S remained

the

S-to-AR

be consistent postulates

systems linking the activation that

to

the

operates

protein while

transition.

This

with the model two sets of intra-

the sperm of protein

zona-induced

in mouse sperm signal transduction

the

had been transition (Endo et

that one set of zona the B-to-S transition

would which

concept

bound

receptor kinase C. acrosome

through mechanism

an intrainvolving

second messengers, having some features in common with those intracellular regulatory systems characterized for hormone-induced actions in somatic cells (Rodbe!l, 1980; Ross and Gilman, 1980; Bernidge, 1984), receives further support from the observation that mouse sperm contain a protein with properties similar to the inhibitory guanine nucleotide-binding regulatory protein, Gm (Kopf et a!., 1986). This protein appears to mediate signal transduction distal to the binding of cell surface receptors by their respective ligands in somatic cells (Gi!man, 1984), and has been implicated in such cellular functions as receptor-mediated (Gomperts, Davidson, cyc!ase 1985).

changes

Ca2

+

mobilization

1983; Moiski et al., 1984; Haslam and 1984; Turner et al., 1986) and adenylate regulation Functional

(Gilman, inactivation

in mouse sperm by inhibits the zona-induced B-to-S hibitory

in

transition effect

5’0(2-thiotriphosphate)

of

stage; lAP

1984; of the

islet-activating acrosome is

protection conferred

(Endo

et

Smigel Gi-like

et al., protein

protein reaction

(lAP) at the

against the inby guanosineal.,

1987a).

The

LEE

626 means provide

by the

which

these

regulatory

elements signals

link

that

ultimately

together

to

mediate

the stages of zona-induced acrosome reaction have not yet been elucidated. Enough evidence is at hand, however, to include the zona-induced acrosome reaction responses signal

in mouse utilizing transduction

sperm a

in the

large

ligand-activated

group

of cellular intracellular

chain. ACKNOWLEDGMENTS

We thank Ms. Cynthia Ward for consultation and organization of ovulation schedules and Dr. Danny Benau for literate and graphic discussions. Excellent secretarial help was provided by Ms. Elvira Walker.

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