in Cumulus: Evidence that the Acrosomes Remain Substantially Intact

BIOLOGY

OF

REPRODUCTION

Binding

31,

of Mouse

in Cumulus:

1119-1128

(1984)

Spermatozoa

Evidence

to the

Z#{244}naePellucidae

that the Acrosomes

BAYARD CAROLYN

T. STOREY,’ MULLER,

and Departments

DAVID

MICHAEL CYNTHIA

of Mouse

Eggs

Substantially

Intact

A. LEE, K. WARD

G. WIRTSHAFTER

of Obstetrics

University

Remain

and

of Pennsylvania Philadelphia,

Gynecology

and

School Pennsylvania

Physiology

of Medicine

ABSTRACT Cumulus-intact mouse eggs bound to the zonae pellucidae observed at 60 mm, after which

inseminated in vitro with capacitated as early as 20 mm postinsemination.

mouse sperm have sperm Maximal sperm binding was time the number of sperm bound per egg decreased to about 30% of binding remained constant from 120 to 180 mm postinsemination. All

of maximal. This extent the sperm bound to the zonae had intact assay. The sperm binding was retarded

acrosomes during

as determined by chlortetracycline fluorescence the first 60 mm by 3-quinuclidinyl benzilate, a induced by zona glycoprotein, but this compound had

specific inhibitor of the acrosome reaction no effect on sperm binding during the next 60 mm. The observation that sperm, which bind to zonae pellucidae after traversing the cumulus, have intact acrosomes is consistent with the same observation of intact acrosomes made with sperm binding to cumulus-free eggs ISaling and Storey (1979). J. Cell Biol. 83544-555l, and further supports the hypothesis that the sequence of events leading to fertilization of mouse eggs is: binding of intact sperm to the zona pellucida followed by the acrosome reaction induced by zona glycoprotein. INTRODUCTION Fertilization

in

collisional

process are

place

in

reaches nisms

the egg plasma of the individual

specific defined

and

the

sequence

processes

comprising

sets

Hartmann,

(Dale

sperm

acrosome

entry of the

the

into

to be:

the

sperm

ago,

Moore this

sperm

the

perivitelline

and

oolemma

sperm

at the

plasma

Williams,

1974).

sequence

are

The

now

postequatorial

membrane first and generally

accepted

pellusperm

which

by

remains

Chang,

Cooper,

the

question

the

rest

before

zona? Huang

from

et

acrosome

on

both (1983)

in

certain

1119

have

before

in

may

vitro

initiate

only zona. in

the

conditions

their

acrosome zona

bound

to

evidence

gametes

that

sperm

the

binding

acrosome-reacted From extensive Chinese

hamster

Yanagimachi that, while fertilizing

acrosome

been But

the

has

presented

occurs

and in vivo, concluded

yet 1983).

the

sperm

pig

fertilization vitro have

not

penetrates

guinea

zona and that can bind to the

work

zoa

does the

and

of penetration

which

(1981)

with

reaction

to the sperm Accepted September 4, 1984. Received March 5, 1984. ‘Reprint requests: Dr. Bayard T. Storey, Dept. of Obstetrics and Gynecology, Medical Labs 339, University of Pennsylvania, Philadelphia, PA 19104.

al.

Wolf

Yanagimachi,

sperm has and Bedford,

or after

studies

1979;

mechanism

sperm

1970;

Yanagimachi,

and

remains: the

a of

1958;

Noda,

and

Phillips

is

Bishop, and

and

1979;

in

and Noda

1983; part

reaction penetration

the

1975;

a

unresolved.

sperm (Austin 1968; Yanagimachi

although

occur

of

Hartmann, It is the middle

for

Overstreet

reaction

obtained from (Yanagimachi,

acrosome

prerequisite

and

the

and of the and

1983a,b; 1983).

by acrosome-reacted worked out (Moore

fusion

(McRorie last parts

species

completed

1982) of

region

sequence

1976;

capacitation;

and

of the

Hamada,

penetration

space;

Bedford, and Bedford,

Niwa

sequence

sperm binding to the zona penetration of the zona;

1981; Moore

zonae Bedford,

1981;

evidence mammalian

necessary

reactions

Monroy,

of

The

definable

these

sperm

reaction;

the cumulus; cida; sperm

of of

1983a,b;

decade

egg: take

the

experimental

variety

The mechaare still largely

and

Bedford,

1983;

Bedford, 1983). A was generally taken

simple

must

before

membrane. reactions

a

and

which

order

unsettled

not

sperm

reactions

unknown remains

is

between

there

a

on

mammals

et al. under spermato-

reactions

on

1120

STOREY

the zona fertilizing Saling

surface, spermatozoa

this

Storey

(1979),

and

have

reported

gametes

from

that

occur

after

the

the

acrosome binding

to

sperm

cannot

carried

rabbit

other

reaction

out

(Bedford,

mouse

appears

the

zona bind

with

1982)

support

acrosomal

is still zona,

in place since the

pieces

thereof

after 1981) system sperm

that sperm receptors

have

the

cap

to the

to the zona are on the

reported by

an

in

to the

zona

with

free,

zona-intact

been

put

we

and

present

sperm

have

intact

The

to

above

cumulus-

criticism

has

would

favor

1983a; Bedford,

Yanagi1983).

fair criticism and addressed experimentally.

report

sperm

the

with

system

is intact (Bedford, 1983; Moore and

paper,

This

bind

sperm and represents from that in vivo where

an eminently needed to be

mouse

eggs.

sperm

to

out

this

of acrosome-intact very different

Storey

intact.

eggs.

that

the

led

carried

mouse

the cumulus machi et al., is

all

and

mouse that

which

forward

binding a system

of

of

its acrosome

were

This earlier

the zona-induced one physiologically

requires

experiments

conclusion

Florman

that the

fertilization in turn

study

vivo

hamster

which

to

conclusion

already bound. with the

led

relevant

In this

(1984)

above

to conclude reaction was

This which

through plasma

Phillips

from

zona

et al. (1980, in the porcine

and

mode

Hall and Phillips,

in sperm conjunction

mentioned

The

binds to the vesiculated

adhering

bind which

primary

or

Peterson evidence

Yanagimachi recently

that

sperm cap

found

penetration. put forward

membrane.

the

when the discarded were

sperm have

that

vivo

reaction results in

(1982b) acrosome

(Gwatkin

1980; and

idea

the

in

hamster

et al., 1976; Phillips and Shalgi, Franklin, 1981) and rat (Shalgi the

that

to

gametes

1972),

to

and

acrosome set of ones

hand,

with

sperm

Studies

from

on

case with conditions.

experiments

acrosome-reacted zona.

is not the under in vivo

ET AL.

a study

of the

cumulus-intact

evidence

that

one

binding

mouse

the

bound

eggs

sperm

do

acrosomes.

bind to zona is through acrosomal ghosts stillin place on the sperm head, which seem to result from

a

preliminary

reaction

stage

occurring

in

the

of

the

MATERIALS

acrosome

vicinity

of

In the

the mouse acrosome

binding

to

of

the

zona

(1980)

on the

pellucida

1983).

experiments,

Florman

that and

sperm

an

making

mouse

and

egg

to

induce

the

sperm

(Bleil

and

Storey

set

(1981,

both

(QNB)

mouse

to

blocked to

zona-intact acrosome

isolated

ited acrosome induced by

intact

reactions addition

eggs

(Florman

results

imply

mouse

egg but

and

that does plays

an

com-

reactions and

in also

sperm inhib-

Storey,

the not

The

in sperm in suspension acid-solubilized zona

of

1982b).

zona

pellucida

just

act

as

active

role

in inducing

These

of

a barrier

(St.

Louis,

obtained reagents, analytical

from obtained grade.

MO). Water for culture media was Flow Labs. (McLean, VA). All other from a variety of suppliers, were of

Media culture

gametes

was

solution

of

the

medium (CM) used in the modified Krebs-Ringer

Toyoda

et

al.

(1971),

handling bicarbonate

containing

of

sodium

(25 mM), sodium pyruvate (1 mM), glucose (5.57 mM), phenol red (23 MM), and BSA, (2% w/v; 20 mg/ml). Medium CM was first prepared without BSA, lactate

eggs. zonae,

Co.

The

cumulus-

zona-intact

Glutaraldehyde was obtained as a 25% aqueous solution from Polysciences, Inc. (Warminster, PA). The compound 3-quinuclidmnyl benzilate (QNB) was the generous gift of Dr. P. F. Sorter, Hoffman-LaRoche Labs. (Nutley, NU). Chlortetracycline (CTC) hydrochloride, bovine serum albumin (BSA) Fraction V, hyaluronidase (bovine testicular), sodium pyruvate, sodium lactate, and cysteine were from Sigma Chemical

of

1 982a)

benzilate of

and

ZP3, inhibSubsequently,

independent

fertilization

bound

bound

sperm

In

of

the fertilization of zona-free mouse mechanism was shown to be the inby QNB of acrosome reactions in

pound

protein

shown mouse

cumulus-free,

but not eggs. The hibition

the

3-quinuclidinyl

the

effects Wassarman

and

glycoproteins

was in

that sperm

a different

the

one of these, designated binding to the zona.

Wassarman,

inhibited

from

Bleil

of

reaction

found

evidence after

wmth

three

glycoprotein

acrosome

come

sperm.

zona

showed that ited sperm

intact

has

isolated

the

further occurs

dealing

protein

this

system, reaction

experiments

zona

up

METHODS

Reagents

cells.

set

AND

cumulus

the to the

then

sterilized

(Nalgene), single use.

by

passage

through

a

0.20-Mm filter aliquots of

and frozen at -80#{176}Cin 20-mI The aliquots were thawed as

added, the medium with 5% CO2 /95%

resterilized air to pH 7.4.

as

needed,

BSA

and

gassed

before,

Gametes

Procedures for the preparation based on those of Inoue and Wolf and Inoue (1976), previously described handling of gametes oil (Dow Corning

of

mouse

(1974,

gametes

1975),

Wolf

and Wolf et al. (1977) have been (Florman and Storey, 1981). All was done at 37#{176}Cundet silicone 200, Dow Corning Corp., Midland,

SPERM

equilibrated with humidity. Spermatozoa MI)

BINDING

5% C02/95% were obtained

TO CUMULUS-INTACT

air at 100% from retired

fluorescence method (Saling and Storey, 1979), as modified by Ward and Storey (1984). The modified method is the following: CTC was dissolved in 500 MM in a chilled buffer of 20mM Tris, 130mM NaCI (TN), and 5 mM cysteine, and the pH was adjusted to 7.8. The solution was kept in a light

Swiss Webster mice (Perfection Breeders, DouPA). Caudae epididymides were excised from one mouse, minced with sterile scissors and submerged in 0.8 ml CM. The spermatozoa were

concentration

and

motility

were

epididySperm

determined

1121

(CTC)

breeder glassville,

allowed to disperse for 2-4 mm, after which mal tissue was removed with sterile tweezers.

EGGS

shielded assay,

with

a

container 5

at 4#{176}C at

of the CTC (37#{176}C) slide, immediately

all times.

solution after

M1

At

the

time

of

was added to a warmed which an equal volume

hemocytometer. Only sperm samples with motility greater than 60% were used. Eggs were obtained from virgin, mature (12-14 wk) Swiss Webster mice (Perfection Breeders). Mice were superovulated with intraperitoneal injections of 10.0 IU pregnant mare’s serum gonadotropin (PMSG)

of CM containing the washed eggs with bound sperm was added with thorough stirring.A coverslip with paraffin wax applied at the corners was then attached. The slides were kept in light-shielded containers at room temperature prior to examination of fluorescence patterns.

The

followed

h under

these

examined

with

36-56

h later

by

10.0

IU

human

chorionic

gonadotropin (hCG). Oviducts were removed 13-16 h after the hCG injection and submerged in silicon oil at 37#{176}C.Swollen ampullae were punctured with sterile tweezers and the cumulus-oophorous mass teased Out into the oil.

Sperm

Binding

Assays

Spermatozoa were allowed to disperse from the epididymides into medium CM for 2-4 mm. The sperm suspension was added to an approximately equal volume of CM to give a final concentration of 1-3 X io#{176} cells/mI. Spermatozoa were incubated at 37#{176}C in CM under oil for 60 mm to facilitate capacitation (Inoue and Wolf, 1975). The suspension of capacitated sperm was added to cumulus-oophorous masses containing at least 30 eggs in CM contained in Lux tissue culture 50-well SAS multi-well plates (Miles Labs., Naperville, IL). The point of addition of sperm to the cumulus-oophorous masses was taken to be the zero

time

point.

At

selected

times,

12.5%

glutaralde-

hyde mixed in 1 M Tris buffer adjusted with HCI to pH 7.4 was added to a final concentration of 0.1%. Cumulus-oophorous masses were immediately placed in

a 1-mi

ronidase complete

solution

to disperse

in 3-5 a wide-bore

with

of

CM

the

containing

10

cumulus

mm. Eggs micropipet

were (internal

cells. washed

mglml

hyaluDispersal was

twice

in CM

diameter

>2

egg

diameters)

to remove loosely associated sperm. In this assay, the manipulations required to remove the cumulus cells and wash the eggs removed all but the tightly bound sperm so that the additional step of centrifugation through a dextran step-gradient (Saling et al., 1978) proved to be unnecessary. Under these conditions, the maximal number of sperm bound per egg was close to unity. The

binding

of

sperm

to

zonae

pellucidae

was

scored by phase-contrast microscopy (X400). This method gives the total number of sperm bound per egg. It is important to note that the phase-contrast assay, in the absence of the acetolacmoid staining used in

earlier

studies

sperm bound to in the perivitelline poor visualization. scored

as

between by

titate

the

space Sperm

decondensed

binding

design

(Saling et al., 1978), the outside of the

excluded

binding

The bound sperm parallel experiments

were not penetration nuclei.

to the zona from

to zonae

scored

only

zona. Sperm scored because of of the eggs was

Intermediate

stages

and decondensation

this

with

assay

in order

acceptable

were to

quan-

constant

a Nikon

The

for

bound

Optiphot

at least

sperm

microscope

2

were

equipped

using excitation at 405 nm (30 and an emission filter which shorter than 430 nm and gave

maximum transmittance 500 nm. The eggs also which tends to obscure

at

wavelengths

show the

longer

intense

than

fluorescence,

fluorescence

patterns

of

the sperm. This difficulty was circumvented by applying pressure to the coverslip to rupture the zonae and allow exit of the intensely fluorescent egg cytoplasm. This method has been shown not to affect the sperm (Saling and Storey, 1979). This method also isolates the sperm bound to the outside of the zona from sperm in the perivitelline space or within the egg cytoplasm and with acceptable

so allows specificity.

quantitation

of

this

binding

RESULTS

Examination of mouse sperm bound to the zonae pellucidae after insemination of cumulusintact

mouse

intact

acrosomes

Paired

phase-contrast

graphs

of

eggs

a

revealed

that

according

to

and

the

sperm

the

had

CTC

assay.

fluorescence

spermatozoon

photo-

bound

to

the

zona

are shown in Fig. 1. The sperm head was bound to the zona on the anterior part (Fig. 1A), as found

previously

(Saling

et

pattern cence

in

al.,

ultrastructural

1979).

The

midpiece

and

cence

a

over

precisely

the

Saling

and

sperm

with

determined

band

of

Storey

fluoreshead and

minimal

fluores-

(1979)

was

shown

by

corresponding

to

intact acrosomes, ultrastructural

separate

as study

et al., 1979). It also was the same designated “B” by Ward and Storey

to capacitated undergone the

mined

bright of the

pattern

completely a

fluorescence

segment,

fluorescence

(1984), using the improved this study; this pattern was

zona

of

postacrosomal

by

(Saling pattern

dark

the

studies

CTC

(Fig. 1B), consisting over the anterior portion

The in

remained

conditions.

with epifluorescence, nm half band-width) blocked wavelengths

specificity.

were scored for intact heads by means of the chlortetracycline

patterns

time in

the both

mouse acrosome course

CTC

sperm reaction. of

sperm

cumulus-intact by

assay

shown

phase-contrast

used

in

to correspond which

had

binding system

to was

and

not

by

the

deterCTC

STOREY

1122

ET AL

FIG. 1. Paired phase-contrast (A) and epifluorescence (B) micrographs of a mouse spermatozoon bound to the zona pellucida of a mouse egg originally surrounded by cumulus cells. The procedures for insemination of the cumulus-intact eggs, subsequent dispersal of cumulus cells, treatment with chlortetracycline (CTC) to produce fluorescence and compression of the egg to remove fluorescent cytosol are described in Materials and Methods. The edge of the zona pellucida is indicated by ZP. The tail of the sperm cell is bent back over the head in this specimen. Note the fluorescence on the anterior portion of the head and the midpiece (the posterior portion of the midpiece is out of the focal plane), with a dark band over the postacrosomal portion of the head. Bar=10

m.

epifluorescence counted

microscopy. the

zona

but

sperm

gave

no

bound

to

lB.

assay None

methods of the

course

the

of

this

acrosome

cence of

The

shown

CTC

which zona

traversed did so

sperm

binding

unbound

study

are

outside concerning

second sperm pattern

shown

acrosome-reacted

of the the

method was bound which B shown in in

Fig.

2. Both

The

in Fig.

of

the

lB. assay,

undergoing

CTC

the cumulus mass and with intact acrosomes. assay

of the

necessity question

criterion

those

sperm

bound Since

removes of

whether

course the

time

observed

in

was

30%

the

effect

In the cumulusreactions are

(Florman system,

at 60

there

to about

present

of binding. sperm-binding mm

only

point,

were

not addressed. cells have a retardation

et al., maximal

mm

(Fig.

a

decrease

maximal

2).

1982). In binding After in

binding,

this sperm

observed

between 60 and 120 mm (Fig. 2). Observations made at 180 mm showed essentially the same

fluores-

By the

was

completed in 20 the cumulus-intact

binding

signs

had

of

on the time free system,

showed all

sperm

egg cumulus

vicinity

was

fluorescence

sperm,

method

the same time-course curve. sperm examined during the

reaction;

pattern the

results gave bound

first

the

information

state of the acrosome. The set up to count only those had the CTC fluorescence Fig.

The

binding

to the

instances by sperm

all

there was densation

count

as

at

120

mm,

of sperm penetration head decondensation. clear evidence in a majority

with

very

few

of eggs as scored At 240 mm,

of sperm head deconof the eggs; this had

SPERM

BINDING

TO CUMULUS-INTACT

longer

Phase #{149} CTC

o 0’

time

C

and 100

E

60

mm

360

assay.

fluorescence important the state

times eggs

times

Time,

90

120

Postinsemination

(mm)

2. Time course of mouse sperm binding to pellucidae of cumulus-intact eggs in medium CM. circles (o) denote scoring of binding by phase-

FIG.

zonae Open contrast

microscopy;

filled

circles

(.)

denote

scoring

of binding by epifluorescence microscopy, using the CTC fluorescence assay and scoring only bound sperm with the fluorescence pattern shown in Fig. lB. Sperm binding is expressed as the percent of maximal binding. Each point represents the mean ± SEM of five replicate experiments,

a minimum of 20 eggs scored in each experiment. Maximal sperm binding scored by phase-contrast microscopy was 1.10 ± 0.15 sperm/egg. (mean ± SEM); maximal binding scored by epifluores-

cence

with

with

become of

CTC was 1.07

maximal

sperm

at

±

agreement

to

observed

cumulus-free densation

system, as penetration

al.,

1983).

This

eggs

that

the

time

zona

to

was

some

end

of

sperm point

the

QNB

As

was

shown

and

mm

was

the

decline

very

QNB 1B)

and

evident.

cumulus

cells

bound to therefore

whose

state

cells

of the

At

had

the

time scale of the 3 is shown in Fig. 4.

intact during progressive

120

mm,

dispersed.

densely

at

under

the zona during passed through

were

Florman

cumulus

postinsemination

remained after which

became

had had

The

and over the in Figs. 2 and

The cumulus 15-30 mm,

dispersal

by

the first dispersal

most

The

of

sperm

the

which

the first a cumulus

30

mm mass

packed.

0M

QNB

5014.M QNB

0’

I00

decon-

(Florman

bound

Fig.

in binding

similar

absence of QNB. was found in the QJ”IB,

times

conditions experiments

in the

head

being

in

3, the

50 iM QNB is to decrease binding times. Maximal binding was still mm

increasing

sperm A similar

examined

further

(1981).

cumuli as that this concentra-

80

et

all

sperm CTC

60

j40.

to

bound fluorescence to intact

the

next

60

in

the

observed

in

the

of

of binding absence of presence

pattern acrosomes.

Time, Postinseminatmon

earlier at 60

observed

over

that

by the effect

at the

The same degree presence as in the

had the corresponding

‘C

0

to 92% in the cumulus-free Storey, 1981), implying

and

cells.

Storey

not

3. that

E

(Florman

cumulus

and

but

2 and

reported

the

in good

previously

using

as compared

that

noted

Figs.

and

the

study.

the by

these studies cells during the

dispersed

course

relative

The effect of 50 iM QNB on the time course of sperm binding was also assessed. At this concentration, in vitro fertilization was shown to be inhibited by 65% in the cumulus-intact system system

was

in in

at

in this

in

acrosomes

increased

marked

compound. times

had

show

effect

penetration seen at 240

sperm/egg.

mm. of

binding

with

1982,

0.18

360

penetration

of sperm

time

was

utilized

longer intact

utilized

insemination

visible the

of the

the had

(1976)

cumulus-intact

tions 60

little

question of the cumulus

Inoue

and

dispersal I-I

30

had

absence

CTC

Wolf

20

in the at still

insemination

‘4#{176}.

0

QNB

All sperm bound presence of QNB An regards

a-

scale,

1123

at 180 mm but clearly inhibited and sperm head decondensation

V .

EGGS

of (Fig. On a

FIG.

(mm)

Time course of mouse sperm binding to pellucidae of cumulus-intact eggs in the presence 3.

zonae of 50 MM QNB. scopy; cence course

the

Scoring was by phase-contrast microa check on selected points with CTC fluoresshowed no difference, as observed in the time determination showed in Fig. 2. Each point is

replicate

of five experiments with and is the mean

eggs per experiment binding was 1.06

t

0.10

sperm/egg

minimum ±

(mean

SEM. ±

of 20 Maximal

SEM).

STOREY

1124 DISCUSSION

ET AL

of

what

occurs

in

The cumulus cells surrounding mouse eggs do provide a barrier for sperm attempting to approach the eggs. The time course of binding

this point would study of the type machi and Phillips

is retarded bound per

be

and the average number of egg is reduced by 2- to 4-fold

pared

to

a cumulus-free

system

sperm

cell

concentration

(Saling

All the sperm zonae had intact

encounter

results

obtained

FIG. (8), mass dispersal

1977)

at

mouse mass

sperm can and reach

space

and

without undergoing the At the earlier times, however, its densely packed character,

telline

in

vivo.

in vitro

We

are

4. Phase-contrast

cumulus

cells

a fair

feel

by

spermatozoa

to the zona resembling, the sperm that

these

representation

micrographs 120

the the

mm with

enter

the

into

the

entry

requires

little with

But

Wolf

entry

occurrence

to

time

the

decreased 120

sperm

had

be

egg at

which begin

found

which

by

(Saling of the

the

per

value

system.

binding perivitelline from should

bound

of

in vitro by Yanagi-

was

after

shown

space

QNB,

validation

period

cumulus-intact

reaction inhibitor

full

binding

mm,

constant

time

but

sperm 60

of sperm

the

60 mm (C), 75 mm (D) and and subsequent insemination of

number

of by

al.,

of at

the outside as assessed

et

and so the sperm which have bound must have traversed a cumulus mass with reasonable fidelity, that which would

same

extent

maximal

essentially

implies that the cumulus

the zona pellucida acrosome reaction. the cumulus retains

the

The

1978).

bound to acrosomes

CTC assay. This penetrate through

at

sperm com-

vivo,

require an extensive done with hamster (1984).

to an

mm. et

This

al.

is

(1976,

perivitelline to

egg the

of the

in the perivi-

acrosome

et al., 1979), and the specific zona-induced acrosome reaction, effect

time space

of

zona

(Fig. 3). If entry into was the route of sperm

on

this

pattern

the loss

zona, maximal binding after maintained in the presence

60 of

mm QNB.

of cumulus-intact mouse eggs in medium CM at 15 mm (A), 30 mm (E), postinsemmnation. The procedures for isolation of the egg-cumulus mouse as time

spermatozoa postinsemination

are

described increases.

in Materials Bar=100

and m.

Methods.

Note

the

SPERM

BINDING

TO

CUMULUS-INTACT

EGGS

1125

1126

STOREY

At present, we binding pattern. cumulus-free

have It

no explanation is not observed

system

implying involved.

that

(Saling

the

et

cumulus

for in al.,

cells

are

this the

and Bedford point that

between

“intact” loss

actual

of

being

overlying

the no

acrosomes,

acrosomal

in which

outer

cap.

the

and

An

plasma

acrosomal

permeability points of

made the distinguish intact

membrane

barriers

in

fully

fusion

with

its

place acro-

outer

membrane. A reacted acrosome has points of fusion with the outer acrosomal membrane, resulting in the formation of pores giving access to acrosomal enzymes, yet the acrosomal

somal

cap

membrane zona. Loss “complete”

may

still

sites of

be

acrosome readily

fluorescence

assay

1979). outside somes when

with

reaction,

which

study bound

CTC

calibrated

of

by

the state of zonae (Saling

to

It was found of the zona cumulus-free

is morThe

originally

was

plasma

binding to the cap leads to a

distinguishable.

an ultrastructural acrosome of sperm the

place

available for the acrosomal

phologically

al.,

in

the et

that sperm bound to had fully intact acroeggs with bound sperm

were recovered at short times postinsemination. The CTC fluorescence pattern corresponding this condition was called Category 1 (Saling Storey, seen in

1979) and was identical to the Fig. lB. Category 2 designated

fluorescent pattern of fluorescence fluorescence head, while bright

fluorescent

and

occurring.

spots

outer from

to correspond to electron microscopy categories

over

bright

to and

pattern a CTC spots on the

portion of a pattern a weakly

the of

fluores-

were taken to be two interof the acrosome reaction in vesiculation of the fused acrosomal

Category

fluorescence

which very superimposed

over the anterior Category 3 designated

cent head. These mediate stages which extensive plasma

in were

4

the loss

was

sperm

membranes complete

head;

represent

the

limits

was loss

it was

of the acrosomal (Saling et al., 1979).

of

shown cap

barriers reaction,

pores formed,

giving access to but vesiculation

Such

differentiation

in in

These

of resolution

acrosomal had requires

place which

and

an small

enzymes had not occurred. the

use

of

for

reactions, (1978),

to

sperm

observed

per-

egg

of

mouse compound

to

Storey,

at

been

It had

sperm which reaction bind pellucidae of

and Storey, 1979). The found to inhibit fertilizaand cumulus-free eggs (Florman and

consistent

being

In this has

system.

cumulus-intact in zona-free

1981),

block

date.

reaction

cumulus-free

(Saling QNB was

in both but not

et al. by the

the cumulus-intact system of acrosome intactness as

in the

eggs

acrosome

by Saling supported

reported binding

the zona

fertilization, by

shown previously that mouse undergone the acrosome poorly, if at all, to zonae

tion eggs,

presently

limits

followed

evidence the

been have very

mouse

zonae

shown to occur in with the same degree was

is

these

originally proposed seems reasonably well

the

with level

the

of

inhibitory

penetration

of

the zona pellucida. This compound was subsequently shown to inhibit the acrosome reaction in sperm bound to the zonae of cumulus-free eggs (Florman and Storey, 1982). This mode of inhibition

in turn

implies

that

bind to the zona in both cumulus-intact eggs must acrosomes, otherwise QNB fertilization

in

Bleil

both

and

these

the

(1980,

both

the

sperm

solubilized

may

One be the

protein

candidate interaction

membrane which

binding The

to zonae results

(Saling

et

al.,

site

the zona which

and

the

not interfere reaction by

(Ward

and

the

induction

for of ZP3

activity

plasma (1982),

have of

acrosome reaction. since inhibition of

(Saling, 1981) does of the acrosome

zona

1984).

and intact inhibit

it is one the ZP3,

binding

activity for inducing the The two sites are distinct, the binding site with induction

which

1983)

shown in an elegant study that three glycoproteins constituting pellucida, which they designated contains

sperm

cumulus-free do so with would not

systems.

Wassarman

syltransferase by

of the CTC fluorescence assay. The assay would not be able to differentiate between a completely intact acrosome with plasma mempermeability acrosome

sequence binding

sperm

study,

has

question

Within

our results are consistent with by sperm with intact acrosomes.

experimental

membrane

this

investigated.

assay, binding

1978), somehow

have must

“reacted”

the

is one

membrane and has

(1983) one

and

acrosome

brane initial

probes;

meability

The

Moore important

four

ET AL.

on

the

with

the

Storey,

site galacto-

mouse

described by Shur again would require

sperm and

Hall sperm

with intact plasma membranes. of this and previous studies 1979;

Saling

and

Storey,

1979)

are at considerable variance with those reported by Huang et al. (1981) for guinea pig sperm. The acrosome reaction in guinea pig sperm is readily acrosomal found

observed cap. to

bind

because of the Sperm in the reacted to

and

fertilize

guinea

size of the state were pig

eggs.

SPERM

This

may

simply

species.

The

be a case

acrosomal

is too small microscopy; designed

zona

to

overcome were

intact it

guinea

pig

events

would

guinea the

zona

the

appears

to

to

acrosomal

species state

at

acrOsome

this have

the

point

of sperm

acrosomes

sperm

and

of attachment

primarily

examined

of the

the

through

(Yanagimachi from a wider

are

for

sequence

detach

mode be

the with

cap

mouse

their

acrosomal ghosts 1984). As gametes

mammalian

the

sperm

between

and

zona

positioned

that to

Hamster

If

the

acrosomal

enough

sperm

by phase assay was

to

undergo

CUMULUS-INTACT

between

limitation.

properly

TO

mouse

this

expected

in size

of and the this

be

pig sperm,

retained Phillips,

of the

bind

then be

But

zona.

intermediate to

and

is large

the

to

would

penetration.

from

of difference

cap

to be accurately visualized the CTC fluorescence

spermatozoon acrosome reaction,

BINDING

with of zona

and variety regard binding

to interaction between zona protein and acrosome reaction, the relation between particular sequence of the fertilization

process

and

should

become

the

architecture

of

the

sperm

head

clearer.

This

dinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa. Exp. Zool. 216:159-167. Florman, H. M. and Storey, B. T. (1982a). Characterization of cholinomimetic agents that inhibit

by

Ms.

Excellent

Neisha

secretarial

service

was

provided

Son.

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