May 4, 1990 - (diethylstilbestrol,. DES) estrogen for the first 5 days after birth results in permanent disturbances in reproductive performance. Ovaries from.
BIOLOGY
OF
43,
REPRODUCTION
Ovarian
(1990)
472-477
Reproductive
Function ANDERS
after
Exposure
HALLING
Department
and
to Diethylstilbestrol
JOHN-GUNNAR
of Anatomy,
University
in Neonatal
Life1
FORSBERG2
of Lund,
Lund,
Sweden
ABSTRACT Inbred
and
after
birth.
1-5 the
in
random-bred At the
ampulla
treatment,
of the
ova
number
were
NMRI
age uterine
found
of ovulated
ova
was
and
a vaginal
plug
grafted
to the
ovarian
ovaries,
never
became
rate
ovaries. indicate
and
litter
size
In the
grafting
associated
experiments,
with
similar
concrements
when and
but
none
females,
became
mice
rate
was
stones)
and
Neonatal
treatment
the first 5 days in reproductive that
were
ized
of female
or synthetic
mice
treated
neonatally
with
in different
ovulation-inducing
stages
thus are not gen-exposed
[51.
ascribed to appear
to postnatal a reduced steroid
have
that
age,
these
ovaries
itself
adult well
females as grafting
females
that had ovaries
causes
the
genesis to become In our previous
5 pg
DES
on
ovary
with
corpora
obtain
Day
[3,6,
in adult
1-5
Day 28, estrosynthetic ac-
corpora
lutea
have
in a dose-related
did we
in females
been way,
observed but
at the
of
neonatally
results].
On
to DES,
some
[7,8]. highest
the
[6,9,
November
‘This investigation Council
(project
no.
30,
DES
1989.
7, S-223
62 LUND,
Dr. John-Gunnar
Forsberg,
Department
of Anatomy,
All deaths
were
fertile
DES-exposed
females ovaries
of locally genotoxic purpose
have may
be
produced steeffects of DES
of this
study
was
to
and oocytes with DES.
METHODS
472
were from closed of the NMRI strain
stocks of obtained
Wiga, Sulzfeld, FRG. The inbred through a strict brother-sister were
kept
in a controlled
humidity) with were fed a standard
en-
a lighting schedpellet diet (R 3;
Sodertalje, Sweden) and given tap water ad was checked regularly for absence of antihepatitis
virus.
of Animals were
cages. the
allowed
Within
pups
were
to nurse
one
treated (Sigma
with
24
one
to give h after
sexed
litter
Chemical
determination. The cause it consistently
Biskops-
Sweden.
study mice
animals
and
culled
daily Co.,
oil or the vehicle only birth. The first injection
was supported by grants from the Swedish Medical Research B90-12X-09056-O1A) and the Crafoord Foundation, Lund, Swe-
2Correspondence:
All
to mouse
female,
pg
den.
gatan
males.
AND
system.
were Received
or control results also for fertility.
main
used in this random-bred
All females
4, 1990.
May
the
mating
allowed Accepted
DES-exposed
of gestation)
from Charles River were propagated
separate
is reduced (100
or
pregnant;
oflpring. The are important
9-16 within
10]. Thus,
Treatment
hand,
well-defined used
control
became
with
in 1987 animals
bodies
tests to 5 pg
other
Fertility dose
Days
AB Astra-Ewos, libitum. Serum
an
fertility
in the
[71.
vironment (temperature, ule of 12L: 12D. They
0.001-
observed
in several
exposed
prenatally
with
or DES-treated
carrying
caged
developing
Animals inbred or
steroido-
treated
we never
birth,
nor
unpublished
exposed
mice
of which
ova
Animals
a steroido-
of ovarian
normal
The
control hosts,
supporting
females. had
ovaries,
MATERIALS
to DES neonatally as females to control
pattern [3, 6]. on adult
after
lutea,
Forsberg, mice
exposed DES-treated
deviating normal studies
a pregnancy
DES
been from
on
ovulations
genic pattern different from that displayed by nonestrogenexposed (control) ovaries [61. HCG or LHRH treatment
gonadotropin
examine the functional capacity of the ovaries from adult females that were treated neonatally
no cor-
exhibit
were
females
observed
Oocytes
character-
and
inbred
Days
females
exposed to an abnormal milieu roids as well as to the potentially
interstitial tissue to a failure of
141.Prior
induced ovaries
After
are
of development,
LH surge
they
After
on
occurred
complications.
for
disturbances adult females
estrogens
from DES-treated
to apparently function, both
DES-exposed
been
(estra-
estrogen
after birth results in permanent performance. Ovaries from
by follicles
tivity
natural
DES)
pora lutea, and hyperplastic-hypertrophic [1, 2,3]. These changes have been the
with
mated.
oil) never
of DES-treated
or DES-exposed
DES/kg/day
(diethylstilbestrol,
not 80%
Ovaries
(olive Ova
DES-treated
ovaries rise
or vehicle ovulation.
were
hosts.
of whether
in inbred
intestinal
mice
pregnant.
thus give endometrial
INTRODUCTION
diol-1713)
these
control
regardless
found
day)
to induce
in approximately
ovariectomized with
to DES can tubal and/or
per
hCG
of gonadotropin-treated,
or DES-treated
for control
(vaginal
percent
5 g
and
females
males
exposed neonatally disturbances, e.g.
(DES,
or eCG
females
Control
mortality
saline
oil.treated Twenty
of control
pregnant.
a high
vaginal
with
caged bursa
were
Oocytes from ovaries DES-induced nonovarlan
groups.
diethylstilbestrol with
DES-treated
of all olive
in both
being
after
with
treated
of saline-treated,
similar
were
treated were
ampuila
females pregnancy
were
females
tube in the
ampuila DES-exposed
mice
of 8 wk,
s.c.
birth birth
every
to their mother
to 8 females. injection
St. Louis,
MO)
of 5 .tg DES genotoxicity
animal
These
in was
litters
containing in 0.025
(controls) for the first was given immediately
daily dose induces
litters
of a random-bred
5 p.g ml
olive
5 days after after sex
was chosen in target
becells
NEONATAL
(Forsberg, roidogenesis
unpublished), [6], and
Inbred
females
DLETHYLSTILBESTROL
a deviating female sterility
were
treated
pattern [3].
of ovarian
identically,
except
The
&periment
purpose
I
of these
to determine
of the
experiments
the
was
exposed treatment
number
if
neonataily to DES could with ovulation and, if
of ova
in the
ampullar
part
tube. Eight-week-old random-bred females, neonatally with DES or olive oil, were given an i.p. of 5 LU eCG (PMSG; Sigma Chemical Co.) at 1600-
1630 h followed hCG (PregnyV, treatment were injection
(or
separately
48 h later Organon). given saline
second with
females
checked
in Bouin’s
solution. and
females
The
serially
sections uterine
then tubes
each
On
the
for vaginal
and DES-exposed tubes and ovaries
paraffin
injection), male.
DES-treated
Control uterine
were were
plugs. was
not
females were were removed
stained studied
F4eriment
To study
the rate
at the hilus other
One
group with
a group
of ova was loss
incidence
number
of females
A.
Untreated
B.
Ovariectomy
plug
with
Experiment I and then were were checked every morning
bursal
litter
with
size
in each
group
struc-
in
our
from
un-
were groups
studied (capin Table 3; in the
same
ta-
females.
(through
the bursal
wall)
of control
fe-
D. E.
from
control
females
grafted
females.
Ovaries tomized Ovaries mized
from DES-treated females grafted to ovarieccontrol females. from control females grafted to ovariectoDES-treated females.
G.
Ovaries
autografted
am-
H.
females. Unoperated
I.
Unoperated
Two
weeks
higher upon After litter
on
DES-treated
after
females
1 female
selected
not
males.
with
caged
from
cage for a period instead of inbred
with
caged
was
randomly
rate. A male was checked regularly;
DES-treated
caged
females
surgery,
males
to ovariec-
grafted
ovariectomized
DES-treated
fertility were
Females
to ovariecto-
control
eosin. for
caged with males. for the presence
litters
is given
from DES-treated females DES-treated females.
and and
the
ovaries. rate
of 21 consecutive
Ovaries tomized
DES
cavity,
grafted mortality
and
F.
formation
neonatally
the
off An-
mized
noninbred
of vaginal
in the
males.
in
in the
made
ble):
ony in a separate males were used
treated
into
NMRI females was used. The following experimental groups ital letters refer to the corresponding
7 p.m);
considered to of ova; thus it
exteriorized
was
inbred
C. Ovaries
II
in females
colony,
was
the body cavity, and the skin-musBoth ovaries were removed from
replaced
for
ovary incision
and the ovary was pinched through the bursal opening.
replaced into was sutured.
breeding
of sa-
in hematoxylin and under a microscope
membrane,
were
treated
the A small
and extirpated was implanted
ovary
males.
embedded
anesthesia, incision.
each female and As a standard
caged
treated in adult life with gonadotropins, a second series of superovulation experiments was performed. Control and DES-treated females were treated with eCG-hCG as in Superovulation The females
bursal
morning,
thickness:
of ova
Superovulation
was
caged
were
(section
part
pregnancy
ovarian
then killed, and the en bloc and fixed
preparations
sectioned
female following
and the total number were counted. This method have no disadvantages and avoided any was preferred to the flushing technique. presence
ether
a dorsal
by another i.p. injection of 5 IU Controls for the gonadotropin only. Immediately after the hCG
saline
a single
were
line-treated
pullar
to determine
uterine
treated injection
under
through
tures were cle incision
adult ovaries from females respond to gonadotropin
the The
inbred feoil as de-
above.
Superotiulation
so,
gen-
473
REPRODUCTION
AND
mice
ste-
that
der was not determined; thus, entire litter of males and males was treated with DES or olive scribed
TREATMENT
males.
with
1
a breeding
or 2 col-
of 60 days. Noninbred males because of their
breeding.
used only for one when definitely
pregnant
visual inspection, males were removed from the cage. that, females were checked daily for birth of a litter, size,
and
offspring.
possible
Every
external
mother
malformations
animal
was
among
allowed
to
the
become
pregnant
only
cage. The females were observed for 25 days for possible delivery. None of the females lacking a plug became pregnant.
Mortality
Studies
During mortality tally with
rate
Grafting
tion, the following experiments were performed. Eight-weekold females that had been treated with DES neonatally were
of vaginal
plugs;
with
a plug
were
put
of adult
whether
living
offspring
females
treated
with
could DES
result
in neonatal
from life
thesia
old inbred
females
grafting
technique
used
were used for grafting experiments. that described by Stevens [11].
The With
our
grafting
among
DES
checked
and exposed to potential genotoxic effects of DES in females with identified ovarian dysfunction [5, 6, 9, 10], the following grafting experiments were performed. Only 8-wkwas
once.
in a separate
Experiments
To determine oocytes
those
and
for vaginal and
then
were
we
experiments,
inbred caged
females males. To
NMRI
with
concrements assigned
while to one
observed
a high
treated find an
under
neonaexplana-
ether
of two
test
anesgroups.
One group of females (n = 22) was caged 1 female with 1 male (noninbred males selected as described in the for were
experiments);
caged
without
females
males.
(n
=
Females
9) of the
that
second
died
during
group
the
474
HAILING
AND
FORSBERG
experiment as well as those that survived after 60 days (the same time period as in the grafting experiments) were autopsied and the vaginal concrements (vaginal stones) were
ond
weighed.
After this no control
Statistics
DES
The Mann-Whitney test for nonpaired samples was to compare litter sizes (Zar, 1984). Chi-square analysis
the
Yale’s
correction
acteristics
was
between
Superovulation Three control males
used
to compare
different
inal able
The plug. ova
latter
of the found
saline and not in the number
caged with of ampullar
of
ova
was
ampullar
had
ova
included
in the
males. ova
among
ama vag-
caged
with
during
males
were
TABLE number
1. Superovulation of ampullar ova
experiments
3.1 young.
males
the
night
control
DES
fe-
and
DES
fe-
after
the
Experiment I: effect of a combined in control and DES-treated females.
are
females
Effect
No. of mice with vaginal plug No. of ovulating mice
No.ofampullar ova per mouse
Saline, not caged with males
from
same later
-
8/20
different grafted
technique used for reto be grafted with other
of the
ovaries
females
with were
X=6.6** (range: 2-13)
treatment
DES-exposed control females 15-24 gave
of the
grafted
from the with control
on the frequency
caged
females (Group days birth,
pups
after and
5/10
13/13 100%
50%
10-19)
used.
had
on 29 grafted
initiation of the mean litter size
any
visible
external
ferwas mal-
of surviving females giving birth was not different from that among with
control
ovaries
(Group
litter size of Group C control ovaries (a mean of 3.9 young
of vaginal
with
males 13/13 100%
(range:
technique
were grafted D); 3 of these
plug
formation,
ovulation
C). Lit-
females per lit-
14.2 (range:
Number of mice with a characteristic trait/total number of mice studied. **Mean number of ampullar ova per mouse calculated from data for ovulating
males
only.
eCG-hCG, Saline,
caged
with
males
with males
caged
4/10 40%
0/35
2/19
8/10
0%
11%
-
mice
and
0/19 0%
-
11-23)
rate,
females
Saline not caged
with
3/10* 30%
15.8
the grafted
ter).
eCG-hCG,
40%
in
ovariectomized
below). All the ovariectomized febecame pregnant, which indicates
control
None
females
females
Saline, caged with males
summarized
were
DES-treated Control
of
ter size was significantly lower (p < 0.001) than that for unoperated control inbred females of Group A (21 females with a mean of 6.5 young per litter) but not significantly
sec-
eCG-hCG
none
formations. The incidence to litters in this test group
or gonadin number
females
control
females (10%) died tility test; 15 females
II control
from
Ovaries ovariectomized
There were no differences between ovulating control
experiment,
size
statistically significant ( < 0.01). The first litter of Group C was born 19 days after initiation of the fertility test, the last one after 51 days. All grafted females in this group survived well.
had no detectof ova in the
gonadotropin-treated
litter
for a mean of 3.9 young per litter. This should be compared with a mean litter size of 6.5 young from 21 untreated control mothers (Group A). The difference in litter size was
two uterine tubes is shown in Table in any of 35 DES females treated with
Experiment
In a second
2).
but No
on other control females (Group C). Of 19 females carrying grafts, 13 became pregnant (68%) and gave birth to a total of 51 young
males.
Superovulation
mean
of grafting
Eighteen
removal
Ovaries
part and
2 with
females, irrespective of saline However, the greatest range
found
results
a complete
saline-treated
with a vaginal plug The mean number
ampullar part 1. No ova were
and DES-treated otropin treatment.
in the 19
females of 8 females
Two females in the ampulla.
The
as described survived and
being caged with (Table 1). None
28
females with a plug gave birth to a litter. (Table
ovaries males 13 eCG-hCG-treated
morning,
had a vaginal for 25 days.
9.7 pups
I all
following
to a litter.
was
the bursal wall (the of ovaries from females
controls had ova DES females, 2 of
group
birth
through moval
10 gonadotropin-treated
pulla.
gave females
RESULTS
of 19 saline-injected DES females, but 4 of 10 gonadotropin-treated DES females had a vaginal plug. When the serially sectioned oviducts were analyzed under a microscope, 5 of 10 saline-treated control females and all 13
8 of
11 of 28 control without a plug
3.
and
the
Experiments
The
females had a vaginal plug after during the night after hCG treatment
For
female control
On
and 3 of 36 DES females females were observed
Table
10 saline-treated
gonadotropin-treated of the oviduct.
treatment. females 2). These
period, females
Grafting
groups.
Experiment
of
used
with of char-
incidences
gonadotropin
of 37 control plug (Table
“5.5 (range:
80%
1-10)
X17.6 (range: 3-27)
DIETHYLSTILBESTROL
NEONATAL
TREATMENT
rol females
Effect
(%)
No. of mice with vaginal plug
28/37* (76%)
No.of
11/37
litters
Litter
size mice
with
To
obtain control
period, vived
49 the
25 days pregnant. When
females
trait/total
(3-60
offspring
from
11 litters
therefore,
variation
were used. Of these (41%), and 29 died
days).
ovaries
None
from
of the
8 of 14 females (57%) died after a days). In another group of 14 un-
caged
females (Group H), none 14 females (71%) died after Finally, none of 15 unoperated
with
males
(Group
I) died
became a mean DES
during
the
num-
surviving
DES-treated
females
Studies
were
in litter
The pected.
size
high There
mortality was no
rate described statistical difference
tween the three groups experiments (Groups nificant (p < 0.001)
a sufficient number of DES females grafted ovaries (Group E) to survive the experimental females test period
not
Mortality
pooled in the protocol; was not estimated.
but (13-53
autografted from one side of each (Group G). None of the females
experiment.
a characteristic
ber of mice studied. **Because of a mistake,
with
0 (0%)
97**
=
of
were side
operated, DES-treated pregnant, but 10 of the of 13 days (4-2 1 days).
3/36 (8%)
(30%)
Number
became pregnant, mean of 28 days
DES-treated females
475
REPRODUCTION
DES females, ovaries female to the other
TABLE 2. Superovulation Experiment II: effect of a combined eCG-hCG treatment on the number of litters and liner size in control and DES-treated females. Cont
AND
above was unexin mortality be-
of DES females used in the grafting E-G, Table 3), but there was a sigdifference in mortality between the
pooled
females
females, 20 surafter a mean of
I, Table exposed
females
3) of 16 DES females observed for 30 days and not to males (no deaths). The results indicated that rate among DES-treated inbred females was the
mortality result of a factor not related to transplantation but more probably to the presence of males.
were
became grafted
on
in these
groups
and
a fourth
group
(Group
or operation Further sup-
DES-treated host females (Group F), none of 14 grafted females became pregnant. However, 7 of the 14 females died 6-23 days after caging with males. To find if mortality was
port for this conclusion was the high mortality rate among intact DES females caged with males (Group H, Table 3). To further investigate the unexpected deaths of inbred
associated with male a graft, the following
on
TABLE
3.
factors, studies
Grafting
operation trauma, or carrying were done. In a group of 14
experiments:
effect
of origin
of grafted
ovaries
DES
females inbred,
on fertility
caged
with
unoperated
males, DES
a special
females.
study
Of
was
22 females
in host females. No. of
Treatment group A
Host
female*
Untreated,
intact
Donor
female
No. of mice studied
litters
21
21/21 (100%)
-
B
Control, ovariectomy, not grafted
C
Control
18
-
Control
size (range)
13/19
DES
29
0/18 (0%) 4.0
=
0/19 (0%)
(1-7)
p Control
-
0
(68%)
D
6.5
=
(3-12)
0/18 (0%)
19
Mortality** (%)
Litter
(%)
15/29 (52’S,)
0.01
