oophorus;. G, granulosa layer; 0. ooplasm; ...... persistently by membrana/cumulus granulosa cell contact. Dev. Biol. 1989;134:297-306. 20. Wassarman. PM.
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
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A, Bleau
G.
Reprod
1987;
ho!
K A glycoprocein
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components
26:599-608.
S, Chapdelaine anna
S, Yanagimachi
Y, Kurata
of
an
36:227-238.
of oviductal
of the
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Uptake
hamster
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Gamete
19:113-122.
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G. Immunocytochemical
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Biol
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RP, Johnson
mouse
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RP, Johnson
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Dev
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Res
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by follicular
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ovum.
the
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in
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In summary,
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development, have been species to cause serious
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of ovulated
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did not change
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