Reversible Inhibition of Fertility in Mice by Passive Immunization with ...

BIOLOGY

OF

43, 385-391

REPRODUCTION

Reversible

(1990)

Inhibition with

JAN

Unites

et 131,

in Mice

Anticumulus

JACQUES

TESARIK,23

187

of Fertility

Institut

by Passive

Oophorus

TESTART,3 National

Antibodies1

GERALD

de

LECAI4

Ia Sante

Immunization

and

et de Ia Recherche

H#{244}pital Antoine-B#{233}cl#{232}re,92141

NOME3

FRANOISE

Clamart,

M#{233}dicale (INSERM)

France

ABSTRACT The

mucifled

definitive

cumulus

expanded

by cumulus

cells.

We

undertaken

was

passive

to

administered

and

human

choriomc

mating

High

recently

doses

antibodies

was

antigens

and

from

reversible

the

did

development

INTRODUCTION Ovulated tecting

mammalian

coat

consisting

of

two

distinct

regulate

coat) specific their

Macek

and

contain different complementary

access Shur

to the

[1],

and Testart [4]). The ments in fertilization nents

potential

oocyte

O’Rand

orus been

intercellular described

matrix

[7, 81. This

Accepted

April

Received

January

17,

‘Support

for

project

search

der

using antibodies into account until effect of antibodies

and

11 this

a Cooperative

latory

velopment

(U.SA.I.D.)

the

rabbit

were of eCG

day

treatment.

The

development.

treatment, findings

These

search

for the

vaccine

most

that,

role of makes

antigens

passive ies on

the egg vesttheir compo-

expansion, present

as

compared part

utilizable

to the cumulus quite recently, to the human

on human discovery

study

immunization fertility in mice. of rabbit

in which We

antiserum

fore and after mating. The cerning the effects of the on oocyte tilization

maturation, development

bility of the was evaluated.

oophorus was when a strong cumulus oophhas per-

we

anticumulus produced

with

analyzed

MATERIALS

pelluovarian

un-

effects

of

antibody-gIobulin

reacting

with

the

(and late preovulatory) eggs mice at different times be-

dose-response anticumulus

effect

the

oophorus a purified

relationship conoophorus antibody

ovulation, fertilization, was examined. Finally,

antifertility

zona

of the

the contraceptive oophorus, we

specifically

cumulus oophorus of ovulated and applied it to superovulated

as targets

in vitro fertilization has opened a new

pilot

less

appearing in the preovu-

with

in a substantial

for

produce

effects if directed to molecules intercellular material only during

cumulus

fraction

antigens

may

dertook

Tesarik

make

appropriate

in theory,

see

[3],

but effect

vaccine.

in the

this

the

before

antifertility

(for

reviews,

on

given

In order to test in an animal model potential of antibodies to the cumulus

recent

serum

(eCG)

preparation

antibodies

on

study

of different

gonadotropin

antibody

of hCG

postfertilization

cida antigens oocyte pool.

of the

AND

anticumulus

and the

postferreversiantibody

METHODS

Antigen Preparation and Production of Crude Antiserum against Ovulated Cumulus-Oocyte Complexes

1990. [CSA-89-045]

Program

Agreement

if applied day

of a contraceptive

serious side the cumulus

1990

Development

affect

the

Compo-

for a future contraceptive vaccine. Since this idea was first formulated [5], a number of studies dealing with various effects of antibodies to zona pellucida antigens have been reported (for review, see Henderson et al. [6]). On the other hand, the possibility of immunological interference with fertilization not taken inhibitory

ovulation

hyperimmune

chorionic the

in its matrix

This

use

that and

[2], Yanagimachi

for

when

on

by

fertility

types of molecules sites on sperm cells

demonstrated control clearly

candidates

anticumulus

a contraceptive

nents, the zona pellucida and the cumulus oophorus, which the fertilizing spermatozoon must first negotiate before it achieves the oocyte cell surface. These egg vestments (the term ‘egg’ is used for the complex of the oocyte and its protecting react with

of this

given

in vitro.

on from

coat

This

of intercellular

fertilization

equine

with

by a pro-

structural

prepared

was

observed

spective

are surrounded

oocytes

human

antibodies

was

and

not

secretion

effect

animals were

antibodies

inhibit

oophorus

negative

antibodies

for

matrix

oocytes.

mammalian

of intensive

superovulated

maturation

oocyte

ovulated

fraction

to mice

of treated

the the

and

candidates

cumulus

of anticumulus

mating

oviducts

when

around as a result

in’imunoglobulin

after

with

found

serious

effects

A dose-dependent the

interfered

were

to the

antibodies

the

layer

development

anticumulus

(hCG),

also

processes

oophorus

that

before

recovered

enveloping

preovulatory

model,

A purified times

an outer

in the

animal

gonadotropin

these

of anticumulus cumulus

shown in an

eggs

antibody

on

effects

late

protocols.

of fertilized

no

have

at different

number

represents

appears

assess,

immunization

and

oophorus

form

was

(CONRAD), with

the

(DPE-3044-A.00-6063-00).

United

provided Eastem

States The

by Virginia Agencr

views

the

Contraceptive Medical

Re-

School,

for

International

expressed

by the

do not necessarily reflect the views of U.S.A.I.D. and CONRAD. 2Correspondence: Jan Tesarik, M.D., D.Sc., INSERM Unite 187, Hflpital B#{235}cl#{232}re, Sers’ice de Gynecologie-Obstetrique, 92141 Clamart, France.

Mature female albino mice were injected i.p. with 6 IU eCG (Chrono-gest; Intervet, Boxmeer, Holland) in 0.1 ml Dulbecco’s phosphate-buffered saline (PBS; Gihco, Paisley, Scotland, UK) between 1600 and 1700 h, followed by 8 IU hCG (Chorulon; Intervet) in the same volume of PBS 48 h

unDe-

authors

Antoine-

385

TESARIK

386

later. cervical

After an additional dislocation and

their large

oviducts. expanded

18-20 ovulated

Eggs consisting cumulus masses

h, animals eggs were

were killed by liberated from

pellucida of other follicles latory, antral follicles with

of oocytes were washed

surrounded by briefly in PBS,

was then absorbed from superovulated

distributed at quantities of 150-200 in Eppendorf containing 0.05 ml PBS, frozen, and stored for 1-3 -40#{176}C.After thawing, 0.05 ml lysis buffer consisting mM Tris HCI here, France)

(pH and

the mixture lysate volume

of incomplete Two male first injected per wk.

1% Nonidet M NaCI was

was emulsified adjuvant (Gibco,

animal and The immune

oviducts

6 and showed

added

La Verpil-

to each

in an equal Detroit, MI)

tube

100 eggs per was assessed

on frozen

unfixed

and

obtained

from

superovulated

and

their

sera

had

anticumulus

(as

titres

with

oviducts 18-20

obtained h after in-

conditions were the same prepubertal ovaries (3 incubaof 4 oviducts each).

from this absorbed by affinity chromatography Uppsala,

hyperimmune

on Protein

(Pharmacia,

diluted with mM Tris-HC1

an equal volume of a solution containing (pH 8.5) and 0.15 M NaCl and applied

Protein

with

20 volumes

ulins

were

eluted

sterilized

by Millipore purity

on

referred

column.

serum

After

was 50 to a

washing

immunoglob-

4). The

eluate

dialyzed against PBS, concen(Amicon Corp., Lexington,

MA).

checked

The

same buffer, bound 100 mM citrate (pH

with

The

Sweden).

A-Sepharose

of the

filtration

of this

SDS-PAGE.

ducing conditions, the ing to immunoglobuhins of

mice

nonpreovuThe serum

A-Sepharose

was

rabbits booster

antibody

homogenates

MA), and Bedford,

above)

Both third

tions

large, but cumulus.

absorption

(4 mg/mI antiserum) was trated by Amicon untrafiltration

of ovaries

mice

respectively. after the

The

absorption

preequilibrated

animal every by indirect

sections

of hCG.

as for the with

including still compact

with homogenized mature female

Immunoglobuhins serum were purified

volume of comfor priming and

then with response

24 h after hCG injection, a good immune response

injection

(Sigma,

Freund’s adjuvant for challenges. New Zealand white rabbits fed ad libitum were s.c. at multiple sites with a preparation of 200

immunofluorescence and

P-40

jection

tubes wk at of 10

was left to stand at 0#{176}C for 15-30 mm. The was adjusted to 0.5 ml with distilled water

and the mixture plete Freund’s

eggs 3-4

7.4), 0.15

ET AL.

Both

typical was

to as anticumulus

(Milhipore

Corp.,

immunoglobuhin

fraction

in nonreducing

pattern observed.

and

re-

of bands correspondThis preparation

is

Ig throughout

the

paper.

>1000.

Anticumulus

Oophorus

The hyperimmune 56#{176}C and absorbed

with

ovaries to remove cept those against oophori gens ries.

during

centrifuged immune

their

serum was homogenates

incubated

not mice

oophorus casional follicles rus

(Table

components of large

1 h, centrifuged

To

1). No reaction Reactivity

mouse

the

could

Unabsorbed Absorbed

were

and

the

twice for

so ab-

specificity

removed

50 mouse

been

removed

of

Antibodies

anticumulus

antibody

cumuli

by absorption with cumulus antigens. Ig (8 mg/2 ml PBS) were mixed oophori from which the oocyte had

mechanically

with

and the mixture was aration of the cumulus

incubated pellet

g and

and decanting was repeated

0#{176}C for 15 mm absorption procedure

fine

hypodermic

of the twice

supernatant, under the

ters

over

the same Ig). with in ti-

1:5.

for an oc-

Passive

with

Mice

the zona

above)

Immunization were

and

with Anticumulus

superovulated

caged

with

as for antigen

males

at the time

Ig preparation

of hCG

of rabbit anticumulus antisera with different ovarian components before and after absorption with ovaries as revealed by indirect immunofluorescence on frozen sections (antiserum dilution; 1: 100). follicles

Nonp reovulato

ry antral

follicles

Preovulat

ory

follicles

0

ZP

G

0

ZR

G

CO

o

ZP

G

Co

-

+

+

-

+

+

+

-

++

+

#{247}++

-

-

-

-

-

-

-

-

+

-

+++

+,

needles

1 h. After sepat 5000 x

at 37#{176}C for by centrifugation

conditions (total of 150 cumuli per 8 mg anticumulus The resulting absorbed Ig gave no specific reaction cumulus materials (assessed by indirect fluorescence)

of preovulatory cumulus oopho-

be detected

Abbreviations: CO. cumulus tivity: -‘ no fluorescence;

action,

were prepared in which specific anticuantibodies in the anticumulus Ig prepa-

of anticumulus

with

Oophorus

cumulus

follicles-except zona pellucida an expanded

were

Samples

ovaho-

the

samples oophorus

ration

anti-

This absorpreactivity with

expanded

Preantral Antiserum

above),

serum. all serum

than

preovulatory with the possessed

(as

test

control mulus

0#{176}C; 2 ml hyperThe mixture was

step was repeated ovaries were used

hyperimmune removed other

TABLE 1. prepubertal

These

of 10 ovaries

x g and the pellet.

was decanted. This of 30 prepubertal

reaction that also

30 mm at mouse

to be present in prepubertal were killed and ovaries were Homogenates

sorption of each 2 ml tion protocol completely ovarian

expansion.

20 mm at 5000 was mixed with

at 37#{176}C for

supernatant that a total

inactivated for of prepubertal

preovulatory

mechanically. for serum

Absoption of Anticumulus with Cumulus Antigens

Purification

all antibodies to ovarian components exthe new antigens appearing in cumuli

were supposed Two-week-old

mogenized

Antibody

oophorus; G, granulosa weak fluorescence; ++,

layer;

0.

medium

ooplasm;

ZR, zona

fluorescence;

+++,

pellucida. Grading of antiserum bright fluorescence.

reac-

(see

injection.

FERTILI1Y

The

males

1100

were

h on the

were

removed

selected.

mg,

from

following

day

Anticumulus

0.5 mg, and

the

and

cages

between

females

with

Ig at three

1 mg)

diluted

INHIBITION

BY

1000 vaginal

different

and plugs

doses

in 0.1 ml PBS was

(0.1

injected

i.p. at three different times according to the superovulation protocol (Fig. 1). In Treatment A, the preparation was

ministered and

3-5

after

h after

eCG;

assessment

C. Control

groups

Anticumulus

was

it

of vaginal

were

created

Ig previously

given

plugs

2-3

for each

absorbed

type

with

hCG B and

of treatment.

cumulus

antigens

group

consisted

experiments, mune rabbit

0.1 ml serum,

mogenized

oviductal

above), were respectively.

given

Evaluation

Fig.

In preliminary and

and

tissues

to

prepubertal

ovarian

experimental

and

Antibody

of anticumulus

effects

B in

8 females.

anticumulus antiserum both previously absorbed

of Anticumulus

Short-term and

of at least

1) were

evaluated

by

preimwith ho-

control

(see

animals,

Effects

on Fertility

Ig on

conception

examination

carefully

removed

fore assessment no dependence of application

with

fine

hypodermic

needles

of oocyte maturity and fertilization. of any of the above parameters on of anticumulus Ig previously absorbed

antigens

a common

was

control

found

are

in control

displayed

teatments,

be-

Since the day with values

in all graphical

of

represen-

tations. Evaluation tility involved ization

(see above) was applied to control animals instead of untreated anticumulus Ig at a dose corresponding to 0.5 mg anticumulus Ig before absorption. Each experimental and control

were

387

ANTIBODIES

cumulus

ad-

h before

in Treatments

ANTICUMULUS

of long-term examination

development

effects on anticumulus of antibody effect

and

assessment

antibody interference with were evaluated by comparing

fertility. the

of the

on

reversibility

of

In both cases, the effects litter size of experimental

and control groups of animals. To examine the effect on post fertilization development, females jected with anticumulus inal plugs and removal reversibility of antibody

Ig on ferpostfertil-

antibody were in-

Ig only after the assessment of vagof males (C in Fig. 1). To assess the interference with fertility, anticu-

mulus Ig was administered either on the day of eCG treatment (A in Fig. 1) or on the day of hCG treatment (B in Fig. 1) and the females were kept in the absence of males

(A

for

30 days.

After

(as

above),

mated,

this

time,

they

were

allowed

and

superovulated

again

to litter.

of eggs

liberated from oviducts on the day following hCG treatment and mating. Females in which mating was confirmed

Statistical

by the

Concepts Inc., Berkeley, CA). Analysis of intergroup and intragroup variance was performed by ANOVA. Means were compared by Student’s unpaired t-test.

presence

dislocation from

of a vaginal

between

prepared

1500

oviducts

and and

plug

were

1700

h. Eggs

examined

microscope using phase contrast. were recorded for each animal: number of mature lated eggs, number cumulus presence

oophorus. of two

killed

were

body

bodies mulus

qualified components

were an

inverted

The following parameters number of eggs ovulated,

ovulated eggs, number of immature of fertilized eggs, and the aspect The distinct

criteria pronuclei

of

classified

ovuof the

as mature.

had

occurred

fertilized. showing

The

absence

and All ferthe first

of polar

an egg as immature. When persisting obscured the view of the oocyte,

cuthey

of

Examination

It FIG.

1 1.

II

,JL U

Day

Application

2

U

Day scheme

3

(A,B)

size .. U

Day 4

of anticumulus

of litter

UU

I U

U

Week Ig in relation

4

to superovu-

lation regimen. In A and B, anticumulus Ig was injected on the days of eCG (labeled PMSG) and hCG treatments, respectively, followed by vaginal plug assessment and examination of eggs on the day following mating. In C, anticumulus Ig was given after mating, and litter size was subsequently evaluated.

Overall Effect on Fertilization The nized

of Anticumulus Outcome

results and

ticumulus

StatView

oviductal

and

protocol

perovulated

mice

serum

Oophorus

of preliminary control sera,

to the same

decrease

IITM

statistical

Antibodies

experiments both absorbed

prepubertal

ovarian

on

the

in the

day

of meioticahly mated mice

anticumulus

with

whole

with

treatment, rate

and

an-

homogeaccording

administered

of eCG

fertilization

with tissues

and subsequently

as compared

Purified

(C)

with

RESULTS

the proportion obtained from

eggs

plug assessment

Examination

II

U

Day

I-Veginal

hCG

undertaken

Chi-square tests (2 X 2 contingency tables) were used to compare frequencies. Correlation between quantitative data was determined as described by Sokal and Rohlf [9].

marked

.1’II

were

(Abacus

fertilization were the and two polar bodies.

PMSG

U

All analyses package

cervical

recovered

under

In a few cases, the first cleavage division the respective eggs were also considered tilized eggs and those unfertilized eggs polar

by

Analysis

to sushowed

an increase

a in

immature eggs in the cohort injected with anticumulus anticontrols

(Table

Ig at all doses

caused a significant diminution eggs recovered from oviducts

of the

2).

used number

in this

study

of fertilized

of passively immunized mice when it was administered before fertilization (Fig. 2). However, this effect was much more expressed when the antibody preparation was given on the day of eCG treatment as compared with application on the day of hCG treatment. A complete elimination of fertilized eggs was achieved with the dose of 1 mg anticumulus Ig injected on the day of

388

TESARIK

ET AL.

TABLE 2. Characterization of the egg cohorts recovered from the oviducts of mated mice iniected on the day of eCG treatment with either anticurnulus antiserum or preimmune serum. No.

No. of

Serum

animals

Fertilized

12 10

7 ( 8) 121 (81)

Anticumulus Preimmune Both

the antic umulus

and

according to the same ‘PB, polar body.

eCG

treatment

(Fig.

tilize mulus

in antibody-injected dispersion, and

were

found

mice had occasional

only

in apposition

that

with

eggs,

and

to their

were

zonae

were mice

examined immunized

of eCG cumulus

injection showed signs of abnormal development with persistence of

cumulus

mass;

these

eggs

usually

lacked

normal corona

cucells Free

of the cumulus the oviducts, toon

a condition found rather frequently undergone complete cumulus dispersion. was a striking reduction in the number

to fer-

pellucidae.

sedimented

petri dishes in which the eggs a few unfertilized eggs from

failed

undergone isolated

cumulus cells liberated after the dissolution intercellular matrix were recovered from gether

sera w ere absorbed

pre immune

with 1 PB

Unfertilized

23 (25) 20 (14)

without

PB

Total

61(67) 8 ( 5)

with horn ogenized

oviductal

91(100) 149 (100)

and prepubertal

ov arian tissues

protocol.

of the eggs

2). Most

Unfertilized

(%) of eggs

the

the

bottom

of

(Fig. 3). Only on

the

day

first

polar

The effect was

time

of administration

but

dependent (Fig.

ment,

on

4).

severely

Ig on the number both

dose

with When

anticumulus it

Antibodies

on anticumulus

eggs regimen

Oophorus

regard

injected

Ig had

impaired

no the

of ovulated

of anticumulus to the on

effect ovulation

the

day

of eCG

body,

ences

from

animals

having there eggs in

reach

statistical

treatment groups. All these factors may have contributed to the observed decrease in the number of fertilized eggs in antibody-treated mice. These findings led us to perform an analysis of separate effects of anticumulus Ig on the number of ovulated eggs, on the proportion of mature eggs in the ovulated cohort, and on the fertilization performance of mature eggs in different immunization protocols. some

12

ovulation

Effect of Anticumulus on Oocyte Maturation When hCG

of mature

eggs

no

Ig was significant

in the

ovulated

of anticu-

of ovulated and the

of control

(Fig.

Oophorus

anticumulus

treatment,

rates

significance

treat-

at the dose of 0.1 mg, rates at the doses of

doses

the

Ig and

superovulation

0.5 mg and 1 mg. By contrast, increasing mulus Ig reduced only slightly the number when applied on the day of hCG treatment,

preovulatory nonexpanded

also in eggs Furthermore, of ovulated

Effect of Anticumulus on Ovulation

eggs differdid

not

4).

Antibodies administered difference egg

on in the

cohort

was

the

day

of

percentage found

as

compared

with controls, irrespective of the dose (Fig. 5). Ovulation of an immature egg was an exceptional phenomenon, since more than 90% of ovulated eggs were mature. However, a quite different situation arose after anticumulus Ig injection on the day of eCG treatment when almost half of the ovulated eggs were immature (Fig. 3), even with the lowest anticumulus Ig dose used in this study (0.1 mg).

Is, v

V*

w

10

a)

8

N

7 6

C) U.4-

0

0

z

U

Day of PMSG DayofhCG

5

4

C

3 2

I ,,

0.1 Anticumulus

0.5 Ig

1.0 Dose

(mg)

FIG. 2. Dose-response relationship of the administered anticumulus Ig to the decrease in the number of fertilized eggs per animal (mean ± SE of 8-12 mice/group) after immunization on the day of eCG (labeled PMSG) treatment (solid bars) or hCG treatment (hatched bars). Horizontal line and adjacent stippled area represent the mean ± SE for control animals. The significance of differences from control is represented as *p < 0.001.

FIG. 3. Immature oocyte (lacking the first polar body) recovered from the oviduct of a mated female treated with 0.1 mg anticumulus Ig on the day of eCG treatment. In spite of the oocyte immaturity, the dissociation of the cumulus oophorus is complete. Note the dispersed cumulus cells (slightly out of focus) sedimented on the bottom of the petri dish. Phase contrast,

x250.

FERTILI1Y

INHIBITION

U

20,

BY ANTICUMULUS

389

ANTIBODIES

DayofPMSG DayofhCG

*

I-i-h U,

LU C)

0.1

0.5

AnUcumulus FIG. 4.

Dose-response

Ig

relationship

0

1.0 Dose

No. of Mature

(mean

anticumulus

±

(labeled

Ig

SE of 8-12 mice/ PMSG) treatment

(solid bars) or hCG treatment (hatched bars). Horizontal line and adjacent stippled area represent the mean ± SE for control animals. The significance of differences from control is represented as *p > 0.05; *sp < 0.001.

Increasing

the

antibody

dose

led

to further

proportion of mature eggs, and mature when 1 mg anticumulus

reduction

of the

all ovulated eggs were Ig was given (Fig. 5).

im-

of the

strong

inhibitory

effect

oocytes

to evaluate

uration

failure

to the

fertilization outcome. positive correlation

the

overall

on anticumulus

contribution effect

lated

eggs

When

the

Ig

mat-

of oocyte

of anticumulus

a)

60

the

number of

of fertilized

mature

number

of fertilized

and

with

0.5 mg anticumulus

injection,

there

ilar was

was

still

a strong

of the respective 6). This correlation Ig dose of of mature eggs

decrease observed

imals

injected

ment

(Fig.

eggs

1 mg from

eggs

=

was

positive

correlation, decreased

not

but progres-

be made

because of the total the ovulated cohort.

in fertilization performance when this correlation was

of mature performed

with

day

anticumulus

Ig on

0.89). plotted

in mice treated with Ig on the day of eCG

functions

could

(r

eggs

ovulated

the

In control animals, there was a strong between the number of mature ovu-

the

of hCG

for the disapA simeggs in antreat-

7).

of Anticumulus on Posfertilization

Lack

Oophorus

a vaginal

Effect

Antibody

Development

anticumulus

senting

plug

Ig was

administered

on

day

the

to animals

following

mating,

prethe

lit-

20

80

I-

and number

eggs and Ig doses

Ig on

100

0, LU

Eggs

number of mature ovulated with different anticumulus

0.1 mg

When

(4

30

Ovulated

against

anticumulus pearance

applied on the day of eCG treatment on oocyte maturation, we analyzed fertilization results separately for meiotically mature

FIG. 6. Correlation between the fertilized eggs in animals immunized on the day of eCG treatment.

the slopes sively (Fig.

Effect of Anticumulus Oopborus Antibodies on Fertilization Pefor,nance of Mature Eggs In view

20

(mg)

of the administered

to the total number of ovulated eggs per animal group) after immunization on the day of eCG

10

U,

Day of #{149} Day of hCG

f..

LU

-c

C) N

40 E

L C)

...+...........

20

U...

.

0.2 Anticumulus

0.4

0.6 Ig Dose

0

......‘

.

0.8

1.0

0

z

(mg)

FIG. 5. Dose-response relationship of the administered anticumulus Ig to the percentage of mature eggs in the ovulated egg cohort after immunization on the day of eCG (labeled PMSG) treatment (stippled line; open points) and on the day of hCG treatment (solid line; full points). Vertical bars and stippled area represent, respectively, ±SE of experimental and control animals.

10

0 No.

of

Mature

20 Ovulated

30 Eggs

FIG. 7. Correlation between the number of mature ovulated eggs and fertilized eggs in animals immunized with different doses of anticumulus Ig on the day of hCG treatment.

390

TESARIK

Mating After Mating

#{149} Before

N U,

C.. C)

-J

0.5 (mg)

0.1

Ig Dose

AL.

sign

FIG. 8. Comparison of the litter size (mean ± SE of 8-12 mice/group) in animals treated with two different doses of anticumulus Ig before (solid bars) and after mating (hatched bars). Horizontal line and adjacent stippled area represent the mean ± SE for control animals. The significance of differences from control is represented as Sp > 0.05; *Sp < 0.001

ment

(Fig.

anticumulus Ig doses the day of hCG treat-

8).

Reversibiliy

of Fertility Antibodies

Oophorus When

same on

mice

Inhibition

previously

by Anticumulus

injected

with

anticumulus

cording to any of the above immunization superovulated again and mated 30 days sistent

reduction

of fertility

control animals. The untreated anticumulus sorbed with 0.9 (mean

±

was

observed

were

Ig ac-

protocols thereafter,

were no

as compared of mice injected

overall litter sizes Ig and with anticumulus

cumulus antigens SE), respectively.

9.8

±

0.8

perwith with

Ig preaband

10.1

This is the first study that demonstrates inhibition of fertility by passive immunization with antibodies against the oophorus.

cessitated previous

a slightly modified strategy study dealing with the

The

nature

of these

experiments

ne-

as compared with our effects of anticumulus

oophorus antibodies on human sperm-egg interaction in vitro [8]. In particular, we had to avoid the use of hyaluronidase for solubilization and isolation of the cumulus intercellular

matrix,

because

the

presence

on the

nents

of ovulated

preponderance

matrix

with

of the respect

cumulus-oocyte

cumulus

to

oop-

other

complexes,

compo-

especially

to

tially present oviductal antigens, previously reported to associate with ovulated eggs of the hamster [12, 13, 14, 15] and mouse [16, 17], were removed by absorption of the hyper-

immune serum tion procedures

of antibodies

indirect

serum

to

this enzyme preparation in hyperimmune sera would compromise their use in in vivo experiments and removal of these antibodies would largely complicate the experimental protocol. Thus, we used the most efficient and the least time-consuming experimental design: preparation of a crude rabbit antiserum against whole cumulus-oocyte complexes, followed by absorption of nonspecific antibodies. This de-

with oviduct homogenates. These absorpresulted in virtually complete (as assessed

immunofluorescence)

reactivity

cumulus

with

oophorus

reaction with was probably

disappearance

ovarian

components

of preovulatory

of

other

follicles.

The

anti-

than

the

residual

zona pellucida of preovulatory follicles to antiserum reaction with cumulus cell secretions, which may impregnate the mouse zona pellucida during the preovulatory period as occurs in human preovulatory cumulus-oocyte complexes [18]. Whatever the nature of this residual zona pellucida reactivity, the effects of anticumulus Ig preparations described in this study were clearly due to a specific action of anticumulus antibodies, since animals injected with anticumulus Ig previously absorbed with cumulus antigens served as controls in all experiments. The global inhibitory effect of anticumulus oophorus antibodies

±

DISCUSSION

cumulus

based

intercellular

by

ter size was not affected by the two doses (0.1 mg and 0.5 mg) tested in this experiment as compared with controls (Fig. 8). This lack of effect contrasted with the considerable decrease in litter size when the were applied before fertilization,

was

horns

the zona pellucida, known to be a strong xenoantigen. In fact, a total of 500 cumuli oophori were sufficient to elicit a strong immune response to the cumulus matrix in the present study, whereas, approximately 10 000 isolated mouse zonae pellucidae had to be used to produce antisera reactive with this egg vestment [10, 11]. Moreover, different types of ovarian follicles-except for preovulatory onesare represented in prepubertal mouse ovaries, thus, these follicles are a rich source of ovarian antigens other than those of the fully expanded preovulatory cumulus mass for absorption of nonspecific antibodies. Antibodies to poten-

C)

Anticumulus

El

the due

on

the

duced

by

crease

in the

yield

several

of fertilized

contributing

eggs factors.

was

obviously

The

pro-

dramatic

de-

number

of fertilized eggs in mice injected Ig on the day of eCG treatment was shown

with anticumulus to be largely due to the antibody effect on oocyte maturation and, at higher antibody doses, on the number of ovulated eggs. The finding of a severe inhibitory effect of anticumulus antibodies on the final phases of oocyte meiotic maturation, expected. meiotic mediated

leading The arrest

to ovulation

cumulus in oocytes

of immature

oophorus approaching

is known ovulation,

eggs,

was

un-

to maintain an

effect

partly by contacts between the oocyte and processes of corona radiata cells traversing the zona pellucida and partly by substances released to the follicular fluid (for reviews, see Wassarman [19] and Racowsky and Baldwin [20]. The inhibitory effect of anticumulus oophorus antibodies on oocyte maturation demonstrated in this study suggests that the role of the cumulus oophorus in the control of oocyte maturation may be more complex and may involve a supporting or permissive action of cumulus components on some events relevant to this process. Alternatively, an-

ticumulus antibodies which cumulus cells

may interfere release their

FERTILITY

INHIBITION

with the inhibitory

mechanism effect on

cyte maturation. Apparently, administration Ig on the day of eCG treatment produces untuning of fine intrafollicular regulatory

different

levels,

abnormal

leading

to decreased

ovulations

cytes.

Apart

releasing

from

these

by oo-

of anticumulus a considerable mechanisms

ovulation

rates

meiotically

effects,

BY

at

and

to oo-

immature

however,

there

of hCG

the

or the

(which

maturity

status

arate

comparison

eggs

in

mulus

immunized

Ig on

the

In contrast same

days

with

after

performance different

or hCG

Ig did

fertilization.

not

The

with final fertilization.

other

affect

thus

sparing

effects.

action

components the

This

antigens,

which

are

that

to their

from

other

reported disturbances

of follicular

with

of the

oping

oocyte

of the

estrous

hibition

pool

after

ticumulus however,

cycle

ultimate

reduction

[21, 22, 23,24,25, [24,27].

The

administration

26]

and

directed

we

reversibility

doses

specffically surrounding

potent

evidence

to antigens

action

and

agents

acting

is perfectly

that

of expanded

preovulatory

contraceptive

contraceptive

here

ovulated

before

includsteroid

oocytes

reversible

after

small

ovarian antigens under

parts structures implicated

investigation

of preovulatory

follides

from destruction. The in this contraceptive in our

laboratory.

and

so sparing

nature effect

ooare This

19.

W.H.

Fertil

cell-

ability:

impli-

of mouse

eggs

in

by antibodies

1989;

Freeman

against

to the

87:193-198.

& Co.;

1981.

eggs

and

zonae

viro

and

in culture.

pellucidae

on

J Reprod

of effect of anna antibody on the development J Reprod Fertil 1982; 66:585-589.

Lack and

in vitro.

evidence Fertil

KI),

for addition

Steril

Chevalier

1975;

properties

of oviductal

of the

pellucida.

A, Bleau

G.

Reprod

1987;

ho!

K A glycoprocein

zona

components

26:599-608.

S, Chapdelaine anna

S, Yanagimachi

Y, Kurata

of

an

36:227-238.

of oviductal

of the

pellucida

Uptake

hamster

origin

egg.

Gamete

19:113-122.

Kan FWK,

s.,

st-Jacques

1989;

Bleau

G. Immunocytochemical

to the anna

antigen

pellucida

for the transfer

evidence

of hamster

ova

after

ovulation.

Biol

40:585-598.

Kapur

RP, Johnson

mouse

ova

LV. An oviductal

and

early

RP, Johnson

embryos.

Dev

LV. Selective

perivitelline

space

fluid

glycoprotein

Biol

1985;

associated

of an oviductal

oocvtes

and

with

ovulated

112:89-93.

sequestration

of mouse

Tesarik

J, Kopecny

oocyte

and

Racowsky

V. Late

cumulus

embzvos.J

Histochemistry Ky. In vitro

arrest

is maintained

fluid

Exp

glycoprotein

Zoo!

1986.

Gwatkin

RBL. Andersen

238:249-

cell

of fertility.

Gamete

Liu C, Dunbar

DM, zonae

Res

BJ, Gwatkin

(Macaca

fasckukis)

reveal

Dev

DI.

Biol

hamster but

oocyte

persistently

1989;134:297-306.

E, NeillJ

1988:

that fluid, (eds.),

The

Physiology

69-102.

Immunization

of female

of anti-anna

with

rabbits

antibody

and

inhibition

1:19-26.

Effect of alloimunization and heteroimmunization on fertility in rabbits. Biol Reprod 1981; 25:439-450. RBL, Yuan LC. Active immunization of Cynomolgus monkeys with porcine zonae pellucidae. Gamete Ret 1983; 4:299-

pellucidae

Gulyas

Press;

production

1980;

studies

In: Knobil

Williams

zonae:

by the human

cxxvte-cumulus-complex

by follicular

contact.

ovum.

the

85:523-528.

in vivo

transiently

Raven

OF,

bovine

Wood with

York:

into

1986;

granulosa New

of proteoglycans

secretion

and

only

PM. The mammalian

Wassarman

synthesis

their

matrices.

heat-solubilized 22.

and

C, Baldwin

of Reproduction. 21.

preovulatory

cells

by membrana/cumulus 20.

24.

other

Skinner

SM, Mills

results

proteins

BS.

T, Kirchick

26.

27.

Sacco

AG,

functional

zona

pellucida

Mahi-Brown

steroid

in squirrel 55000

secretioi

macromolecule.

Biol

B. Nelson immunized

prod

Microbiol

1988;

the bitch adjuvants Reprod

CA, Yanagimachi by active and 1985;

immunization

patterns 32:761-772.

Endocrinology

(Saimiri

of bitches

of estradiol

with 1984;

Reprod porcine

115:2418-2432.

1987;

with

procine

36:481-490. H, Mumbo

zonae

re-

WR. Ovaries

immunized

ML, Yanagimachi

pellucida

and inhibition

EC, Dukelow

sciureus)

with

zona

differentiation

MG, Yurewicz

histopathology Mahi-Brown

Immunization

follicular

monkeys

CA, Yanagimachi

lmmunol

BS.

ovarian

DL, Subramanian

Pierce

main

HJ, Dunbar

in an abnormal

of gonadocrophin.induced 25.

an-

of cumulus is currently

of cumulus

307.

tibody administration. These properties make the preovulatory cumulus oophorus a strategic site for immunocontraception, rendering possible a very specific impact involving only

18.

23.

a single

to the

260.

antibodies

fertilization.

role fertilizing

in vitro

J Reprod

York:

by the hamster

1, Sendai

in the

of an-

cumuli

Roberts

biochemical

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in-

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Effect

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intercellular

devel-

of fertility

of contraceptive

present

17.

abnormalities

function after more prolonged antibody exposure, ing a detailed examination of ovarian histology and hormone production.

phori

in

Ig is encouraging; further studies will be needed, to assess the normality of the reproductive system

In summary,

16.

for

development, have been species to cause serious

Shivers hamster

Reprod

to zona

candidates

embryos

of an oviductal

antibody

antibodies

of mouse

Oikawa

Endocr

54:233-237.

Y, ittingham

Ret 15.

MC.

Tsunoda

Litveillit

D, Veselsky

matrix.

and development

Fox U,

4:729-741. Acta

potential

of human

of human

FJ. Biometrv.

Y, Chang 1978;

1989;

fertilization.

l988;3:129-l32.

intercellular

RR, Rohif

Tsunoda

alters

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9. Sokal

14.

side

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83:325-343.

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1988;

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with

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in the

in !VF. Hum

8. Tesarik

13.

MJ, Aitken

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194:223-244.

J Reprod

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Hum

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CJ, Hulme

7. Tesarik

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1974;

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to the

ef-

undesired

potential

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zona

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when

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preovulatory

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size

ga-

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3. Yangimachi R Mammalian fertilization. In: Knobil F, NeillJ (eds.), The Physiology of Reproduction. New York: Raven Press; 1988:135-185. 4. Tesarik J, Testart J. Human sperm-egg interactions and their disorders: implica-

cation

of antibody

only

Res

fertilization

indicates

the

follicle

of the

is of particular

pellucida

than

ovarian

aspect

of

litter

absence

phases of egg preovulatory Moreover, the antibodies

ovarian

mete

treatment.

fect on postfertilization development inhibition by these antibodies is due ence with

doses

1. Macek MB, Shur B!). Protein.carbohydrate complementarity mete recognition. Gamete Res 1988; 20:93-109. 2. O’Rand MG. Sperm-egg recognition and barriers to interspecies

number

by the sepof mature

and

considerable antifertility antibodies injected before

of anticumulus

administered

either

oocytes),

with of eCG

the

oophorus

doses

of ovulated

of the fertilization

mice

ticumulus

did not change

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treatment

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