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Interference with early pregnancy in rats by estrogenMechanism of action of dienestrol.

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Interference with Early Pregnancy in Rats by
Estrogen: Mechanism of Action
of Dienestrol'
M. J. K. HARPER
Imperial Chemical Industries Ltd., Biological Research Department,
Pharmaceuticals Division, Alderley Park, Maccbsfield, Cheshire,
and the Worcester Foundation f m Experimental Biology,
Shrewsbury, Massachusetts 2
ABSTRACT
Rats were given single doses of an orally-active estrogen by mouth on
days 1-5 of pregnancy. Examination of uteri on day 8 for implantations revealed
that the median effective doses for preventing 50% of the eggs shed from implanting
were 0.047, 0.050, 0.035, 0.145 and 0.380 mg/kg for days 1, 2, 3, 4 and 5 respectively.
It was found that failure of pregnancy after treatment with the minimal 100%
effective dose (EDIw) on days 1, 2 or 3 was due to expulsion of eggs from the reproductive tract by day 5. Treatment of rats with the ED100 on day 4 caused expulsion
of some eggs from the tract by day 5, but also had effects on the uterus: the position
of the blastocysts within the lumen was abnormal and their orientation relative to
the mesometrial-anti-mesometrial axis disturbed. No decidual reaction was observed on
day 6 in such rats or on day 9 in pseudopregnant rats treated similarly and subjected
to uterine traumatization on day 5. Treatment with ED100 on day 5 had no effect
on egg transport or on the positioning of blastocysts within the lumen on day 6.
A decidual reaction was seen around the blastocyst on day 6, but in pseudopregnant
rats treated similarly and subjected to uterine traumatization on day 5 no decidual
reaction was found on day 9. The pontamine blue reaction was negative in nearly all
rats dosed on day 4 and examined on days 6-8. In rats dosed on day 5, however, the
blue reaction was negative on day 6, but positive on day 7, i.e., delayed by 24 hours
compared to controls and appearing later than the decidual reaction. The blastocysts
were unattached in these rats on day 6, but may have attached briefly on day 7
(suggested by the positive blue reaction) and subsequently failed to develop further
owing to disappearance of the decidual tissue.
As early as 1926 it was reported that
administration of follicular extracts would
interrupt pregnancy in mice (Parkes and
Bellerby, '26) and rats (Smith, '26), and
that as time from ovulation increased
more extract was required to produce the
same effect. From Smith's results ('26) it
can be calculated that in terms of estrogenic activity, some 1.2 rat units were required for a median effect (termination of
pregnancy in half the rats) following administration on day 2 and 1.7, 5.2, 8.2 and
21 rat units respectively, when the extract
was given on days 3, 4, 5 or 6.
Dreisbach ('59) showed that lower doses
of estrone were required to interfere with
pregnancy in rats when given during the
period of tubal transport. Edgren and Shipley ('61) found median effective doses of
13, 17, 4.2 and 45 pg/rat for estrone given
subcutaneously on days 1, 2, 3, and 5 of
pregnancy respectively. Saunders ('65) reANAT. REC., 162: 433-452.
ported similar results for estrone in rats
showing that it was most effective if given
on day 3 of pregnancy.
This paper describes a quantitative study
of the effects of an orally active estrogen
(dienestrol) on early pregnancy in rats
and the reasons for the variation in response to estrogens according to the time
at which they are given.
MATERIALS AND METHODS
In most experiments rats from the random-bred colony of specific-pathogen-free
albinos (Alderley Park Strain 1-Wistar derived) were used, but for later experiments
Wistar Strain rats were purchased from
Gofmoor Farms, Westborough, Mass. Virgin females (ca. 200 gm body weight) in
pro-estrus were caged overnight with fer1This investigation was supported in part by a
USPHS Research grant €ID-02597 from the National
Institute of Child Health and Human Development.
Present address.
433
434
M. J. K. HARPER
tile males or with vasectomized males.
Those which on the following morning had
fully cornified smears with spermatozoa
present were assumed to be pregnant (day
1). Those animals which showed a cornified smear after mating with vasectomized
males, were assumed to be pseudopregnant. On various days of pregnancy or
pseudopregnancy the rats received single
graded doses of dienestrol (Boots Pure
Drug Co. or Sigma Chemical Co.) by
mouth. The dienestrol was dispersed in
0.5% aqueous Tween 80, each rat receiving 0.5 ml/lOO gm. body weight.
The procedures followed in individual
experiments ( 1-5) were as follows :
1. Groups of rats were treated with a
range of single doses of dienestrol on days
1-5 of pregnancy. Corpora lutea and implantation sites were recorded, and the
mean diameters of the latter measured at
autopsy on day 8.
2. Groups of pseudopregnant rats were
treated on days 1, 2 or 3 with single doses
of dienestrol known to be the minimal effective on that day for preventing pregnancy (EDlao).On days 4 and 5 groups of
rats received various doses. On day 5 a
sterile silk thread was placed lengthwise in
the lumen of one uterine horn, while the
other remained untouched. The rats were
killed on day 9; the traumatized and untraumatized horns were dissected out, blotted and weighed separately, and sections
taken for histological examination.
3. Groups of mated rats were given, on
days 1, 2, 3,4 or 5 of pregnancy, the minimal doses of dienestrol, known to prevent
pregnancy completely when given on that
particular day (EDloo).Eggs present in the
oviducts and uteri were recovered and
counted, and their condition recorded on
days 5, 6, 7 or 8.The eggs were examined
fresh, and after fixation in 10% buffered
formalin and staining with lacmoid (Yanagimachi and Chang, '61; Marston and
Chang, '64). Photomicrographs of blastocysts were taken in the fresh state.
4. Groups of four or five pregnant rats
were given vehicle or dienestrol on days 4
or 5 (EDloo)and then killed on days 6, 7
or 8 ten minutes after an intravenous
(i.v.) injection of pontamine blue (0.5
ml/rat of 1.0% aqueous solution). The
uteri were inspected for blueing or the
presence of blue patches denoting implantation sites (I'sychoyos, '60).
5. Two groups of three rats were given
dienestrol ( 1 mg/kg p.0.) either on day 4
or on day 5 and killed on day 6 of pregnancy. Two control rats were also killed
on day 6. The uteri were removed, trimmed
and k e d in Alcohol-Formalin-Acetic acid
fkative. After clearing and mounting they
were serially sectioned at 6 p, stained with
hematoxylin and counterstained with eosin.
The sections were then examined for the
presence of blastocysts in the uterine lumen and phaitomicrographs taken.
RESULTS
1. Table 1 shows that the dose of dienestrol required to prevent 50% of the
eggs shed from forming implantation
swellings in the uterus (EDso)was lowest
when the coimpound was given on day 3
of pregnancy. As the percentage of eggs
producing implantation sites decreased after increaslig doses on any one dayso also did the proportion of rats remaining pregnant. In addition, the number of
implantation sites per pregnant rat was
significantly reduced, indicating that the
effect of dienestrol upon implantation in
the individual rat was not a quantal response. The mean diameter of the implantation sites (measured at autopsy on day
8) decreased significantly with the increase
in the dose of dienestrol for each day of
dosing (fig. 1). The effectiveness of dienestrol in preventing implantation was directly relatedl to reductions in these parameters.
The ED50 for dosing on day 3 was significantly difFerent from those for days 1,
2 or 4. There was no significant difference
in ED50 between days 1 or 2, but both these
were significantly smaller than that obtained after dosing on day 4. No comparison could be made with EDSOfor day 5,
since the slope of its dose-response was
much steeper than those of all other
groups. The slope of the dose-response line
for day 4 (a) was, by contrast, very flat
with large 95% probability limits (i.e.,
1.81-6.32-2212). It can be seen (table 1)
that the effects of treatment on day 4 on
both the proportion of rats pregnant and
the percentage of eggs forming implantation sites, were quite unlike those after
10.7f0.5
6.421.4
0.9 -C 0.5
0
0
11.92 0.4
12.3 f 0.9
12.22 0.8
12.5f 1.6
10.3f 0.5
1
2
At autopsy on day 8.
95% confidence limits.
12.0 -t 0.6
3.5 2 0.5
0
0.380
( 0.333-0.433)
92
15
0
12.020.6
1.8-t 1.0
0
13.0f 0.4
11.8 k 0.8
12.82 1.0
4
2
0
123
4
4
4
214
0.25
0.5
1.0
Total
5
5.9 2 1.0
6.7 f0.8
3.02 0.6
1.0
13.2-t 0.7
10.7-C.0.7
11.2 k 0.5
11.7-C0.5
7
6
4
1
10
10
11
11
0.1
0.25
0.5
1.0
4(b)
10.3f 0.2
7.3 f0.9
7.0f 1.1
6.8" 1.7
0
8.4f1.8
5.3 -c 0.8
5.0-t 2.0
0
0
0
0.145
( 0.076-0.276)
0.035
(0.031-0.044)
31
37
10
1
76
50
44
28
63
35
10
0
0
10.7f0.5
8.3 f 1.0
3 . 0 1 1.0
0
0
9.9-CO.6
6.2 f 1.2
4.5 f2.0
0
0
11.6f 0.5
Implantation
sites per
pregnant
rat
(mean 5 S.E.)
4.1 f1.1
4.02 1.2
1.1e0.5
0.1
4
8
8
5
0
4
10
10
10
4
0.05
0.1
0.25
0.5
1.o
4(a)
0
0
7
0
0
0.050
(0.044-0.057)
0.047
(0.042-0.053)
ED503
mg/kg
10.3-C 0.2
6.22 1.3
5 . 6 f 1.3
3.4 rt 1.4
0
13.5 f 2.0
12.3f0.7
12.7* 0.5
12.1f0.3
10.0 f0.9
9
9
2
10
10
10
4
3
0.025
0.05
0.1
0.25
0.5
3
0
0
12.0-C0.5
13.82 1.0
10.5f0.6
12.5-Cl.3
12.3+- 1.5
10
7
3
0
0
10
9
10
4
4
7.62 1.4
4.8 f 0.9
1.0-0.7
9.9 -C 0.6
5.6-C 1.2
1.8 2 1.0
0
0
12.4 f0.4
11.4 f 0.4
12.2f0.6
12.02 0.7
12.3f 0.7
90
52
79
49
15
0
0
11.6f0.5
11.7 t0.5
0.025
0.05
0.1
0.25
0.5
9
9
4
0
0
9
10
10
4
3
0.025
0.05
0.1
0.25
0.5
1
99
Implantation
sites
Corpora
lutea
Per cent
of eggs
fonnmg
implantation
sites
Mean no. (k S.E.)
per rat mated of
2
12
12
0
2-4
Mated Pregnant 1
mg/kg
(P.0.)
No. of rats
D~~
Dosage
inhibition of pregnancy in ruts: Effectiveness of dienestrol given in a single oral dose on different days of pregnancy
TABLE 1
436
M. J. K. HARPER
I
1
j
0025
Dose
of
Control
\
\
05
Dienestrot
025
01
lrng/kg
10
05
b y gavogel
Fig. 1 Effect of single oral doses of dienestrol
on the mean diameter of the implantation swellings at autopsy on day 8.
treatment on days 1, 2 or 3. In order to
determine whether this was a chance result, a further experiment (4b) was carried
out, which produced similar results. It was
concluded therefore that dienestrol produces a type of response when treatment
is on days 1, 2 or 3 which differs from
that after dosing on day 4 or on day 5.
2. The effects of the minimal EDioo..of
dienestrol on the deciduomal response of
the uteri of pseudopregnant rats was examined. Only treatment on day 1 failed to
reduce the deciduomal response to trauma
to a minimum, and this was due to one
rat which responded normally. EDlooon all
other days reduced the ratio T/U almost to
unity (table 2). Examination of histological sections ad uteri from the rats given
dienestrol (EDioo)either on day 4 or day 5
and killed on day 9 showed no evidence of
a deciduomal response to trauma on day 5.
This must be contrasted with the small
decidual response seen around the blastocysts in rats given dienestrol on day 5, but
killed on day 6 (see section 5 ) .
Since similar responses were observed
after doses of 0.25 mg/kg on days 2 or
3, and of 1 mg/kg on days 4 or 5, it was
felt that lower doses at these later times
might also be effective. Thus a range of
doses, with lmown effects on pregnancy
(see table l), was given to groups of rats
on day 4 or day 5 of pseudopregnancy. It
is apparent that the deciduomal response
was reduced very sharply by a dose of dienestrol which did not prevent considerable
numbers of eggs from implanting. When
0.25 mg/kg of dienestrol was given to
pregnant rats; on day 4 or day 5, 44 and
92% respectively of the eggs shed produced uterine swellings. However, the deciduomal response (ratio T/U) of the
uterus of pseudopregnant rats given the
same dose on the same days was reduced
to 33 and 32% respectively of normal.
Thus there 'was no good correlation between failure of implantation and lack of
deciduomal response, since the latter de-
TABLE 2
Effect of dienestrol on deciduomal growth i n pseudopregnant rats
Ratio
Dosage
No. of
rats
0
0
6
Weight of uterine horns (mg)
Traumatizedl
untraumatized
uterine
Traumatized
mean
S.E.
+-
Untraumatized
mean +. S.E.
957.02 36.5
122.8-t 6.6
7.9% 0.5
2.7zt 0.9
1.52 0.2
1.320.3
horns,
mean
+ S.E.
1
2
3
0.25
0.25
0.25
5
5
5
372.82 141.9
231.6-F- 6.7
197.2C 18.1
132.8k 7.5
158.1& 13.5
154.4& 13.8
4
0.05
0.1
0.25
0.5
1.0
4
6
6
6
5
580.8& 98.0
565.8-C-83.5
385.52 75.0
243.4-C-41.3
190.02 17.2
139.5C 9.0
147.31+17.5
145.8212.2
141.02 9.6
131.4-t 6.9
4.1-C 0.5
4
4
4
4
5
5
5
0.25
0.5
1.0
5
4
4
394.6-F-39.9
296.92 40.1
222.02 8.7
163.2f 19.7
206.9f28.8
iwsrc- 7.2
2.5f 0.4
1.52 0.2
1.4k 0.1
4.22 0.9
2.6f 0.4
1.720.2
1.520.2
437
MECHANISM OF ESTROGEN ACTION O N EARLY PREGNANCY
creased more sharply than the former with
increasing doses of dienestrol.
3. From the uteri of control animals
72% of the eggs shed were recovered on
day 5 of pregnancy (table 3); these had developed to the late morula, or blastocyst
stages. On the morning of day 6 only 47%
of the eggs were recovered. By day 7, no
eggs could be flushed from the uterus, and
obvious implantation sites were present.
Administration of the EDlooof dienestrol
on days 1, 2 or 3 of pregnancy accelerated
tuba1 transport and caused premature expulsion of the eggs from the uterus (table
3 ) . Only three eggs were recovered from
15 animals in these three groups. In animals dosed on day 4, however, the situation was different. Although they received
four times more dienestrol than those dosed
on days 1, 2 or 3, 47% of the eggs were
recovered from the uterus on day 5, and
51% on day 6. By day 7 only 10% could
be recovered. Since dosing on day 5 was
done at 1700 hours, expulsion of blastocysts from the uterus seemed unlikely. The
percentage recovered on day 6 was similar
to that recovered from the uteri of control
animals on day 5, but much larger than
that from controls on day 6 or from animals dosed on day 4 and killed on days 5
or 6. On day 7 the percentage recovery
(47) had fallen to the level seen in con-
trol animals on day 6, or animals dosed
on day 4 and killed on days 5 or 6. By
day 8 only 2% of eggs shed could be recovered. In none of these dienestrol-treated
animals killed on days 7 or 8 were implantation swellings visible.
4. In groups of rats treated with dienestrol on days 4 or 5 and given pontamine
blue i.v. just before autopsy, the uterine
responses were different from those of
vehicle-treated controls. In controls a slight
generalized blueing of the uterus was visible on day 5 of pregnancy, and by day 6
discrete blue patches denoting the future
implantation sites could be seen. Only one
of ten rats given dienestrol on day 4
showed blue patches in the uterus on day
6, and none of the rats similarly treated
but killed on days 7 or 8 showed any reaction. Dienestrol administration on day 5
produced initially a pattern similar to that
seen in controls, but delayed by 24 hours.
In most rats generalized blueing of the
uterus was seen on day 6 and blue patches
were visible on day 7. By day 8, however,
when implantation sites should have been
visible, no blue reaction of the uterus
could be seen.
The appearance of the blastocysts
flushed from the uteri on day 6 vaned considerably. Some (fig. 2) resembled normal
day 5 blastocysts after the shedding of the
TABLE 3
E f f e c t of single oral doses of dienestrol o n site o f recovery and stage of development o f eggs f r o m Tats
killed on days 5 to 8 o f pregnancy
Dosage
Day mg/kgl
Dayof
autopsy
No.of
rats
(P.0.)
4
4
5
0
0
0
1
2
3
0.25
0.25
0.25
5
4
4
4
1.0
1.0
1.0
5
5
5
1.0
1.0
1.0
1
7
14
5
Eggs
Recovered
Shed
Total
Stage of development
in
inoviduct
uterus
no.
no.
no.
no.
98
197
60
71 72
85 47
0
0
0
0
-
71
85
0
4
0
1
-
2
-
0
3
0
%
A k y - Morula
no.
no.
Blastocyst
no.
0
17
54
0
0
85
(53 implantation sites)
3
0
0
-
5
5
5
63
71
62
5
6
7
4
13
8
53
192
97
25 47
98 51
10 10
0
0
0
25
98
10
2
1
0
4
0
0
19
97
10
6
7
8
9
8
10
90
86
130
57 63
40 47
2
3
0
0
0
57
0
0
1
0
57
40
2
5
Minimal 100% effective dose on that day for preventing pregnancy.
40
3
0
0
438
M. J. K. HARPER
TABLE
4
Reaction of the uterus to intravenous injection of pontamine blue 10-15 minutes before
autopsy i n relation to day of dienestrol treatment and day of autopsy
No. of rats
Dosage
D~~
mg/k
(P.0.)
Day of
autopsy
With uterine reaction to dye as follows:
with
Injected
dye
Negative
pz&
Slight
geb;E1
color
4
0
1
0
3
0
0
9
0
52
5
7
4
0
1
a
10
7
4
4
0
6
8
7
7
6
2
2
6
6
0
0
0
0
5
0
4
6
7
4
1.0
1.o
1.0
5
5
5
1.0
1 .o
1.0
4
1
2
3
10
5
5
4
6
7
8
0
0
0
5
0
Site of blastocyst attachment.
Implantation sites visible.
zona, while others showed varying degrees
of protrusion of cells or blebbing with or
without cytoplasmic inclusions from the
outer surface of the trophoblast (figs. 3,
4, 5). One unusually large blastocyst was
also recovered from a control animal on
day 6 (fig. 6). Blastocysts recovered on
day 6 from rats given dienestrol (1 mg/kg
p.0.) on day 4 showed a similar phenomenon. The blebbing from the trophoblastic
surface ranged from only 1 (fig. 7),
through a few (figs. 8 and 9) to a large
number of cells (fig. 10). This blebbing
was also seen in blastocysts recovered on
day 6 from animals similarly dosed with
dienestrol on day 5 of pregnancy (figs. 11,
12, 13). Many of these protrusions appeared to contain no cytoplasm and such
blebbing may have been an artefact caused
by the recovery medium (Ringer's solution). This, however, is unlikely since the
later the blastocysts were recovered the
more of them showed this phenomenon
and the more accentuated it became. Since
there were no obvious differences between
blastocysts from control and treated animals, it was surmised that this phenomenon might represent the first stages of
attachment of the blastocyst to the uterine
epithelium, and that flushing the uterus
had disrupted this attachment.
5. To investigate this possibility serial
sections were made of the uteri of rats
dosed with dienestrol (1 mg/kg) on day 4
or day 5, and of control rats killed on day
6 of pregnancy.
In the control rats killed on day 6, 33
out of 34 blastocysts were positioned near
the anti-mesometrial end of the lumen
(figs. 14, 15). Their orientation was uniform since the abembryonic pole was directed into the anti-mesometrial cleft. The
blastocysts were generally elongated and in
some cases appeared to be lying free in the
lumen. The shape of the crypt in the epithelium showed that this was a shrinkage
artefact and that the blastocysts had been
closely pressed against the epithelium
which was still intact (figs. 16-19). Signs
of attachment of the blastocyst to the epithelium were present at the abembryonic
pole (figs. 16-19>. The epithelium in the
region of the implantation chamber had
significantly increased in height from the
level of the embryo to the extremity of the
anti-mesometrial cleft, and there appeared
to be secretion into the lumen at the area
of blastocyst attachment (figs. 18, 19).
The lumen o f the uterus in all sections appeared quite straight and despite a certain
amount of fixation shrinkage was relatively narrow (figs. 14, 15). A marked decidual reaction had also commenced around
the region of increase in epithelial cell
height (figs. 14, 15).
There were obvious differences in the
uterine sections of rats killed on day 6 that
had been given dienestrol on day 4. There
MECHANISM O F ESTROGEN ACTION O N EARLY PREGNANCY
was an increase in the complexity of the
lumen with the appearance of many short
and sometimes branching lateral folds
(figs. 20, 21, 22). In addition the blastocysts were rarely found at the normal implantation site. In most cases (15 out of
21 blastocysts) they were found in the
center or only partially towards the antimesometrial end of the lumen (figs. 2022). Not only was location within the
lumen abnormal, but orientation in relation to the mesometrial-anti-mesometrial
axis of the uterus was also disturbed. Five
of twenty-one blastocysts had their embryonic pole directed towards the antimesometrial surface (figs. 21, 23), 7 had
their polar axis at right angles to the axis
of the lumen (figs. 22, 24) and only nine
were correctly oriented (figs. 20, 25). In
no case did it seem that attachment had
occurred even when the blastocyst was
pressed against the epithelium in a crypt
(figs. 23-25); all the epithelial cells were
increased in height compared with those
of control rats, and there was no specific
reaction to the presence of the blastocyst
(figs. 23-25). However, as in the control
rats the nuclei of the epithelial cells were
positioned on the basal membrane (figs.
23-25). There was pseudostratification
of the epithelium (figs. 23, 24) which was
not observed in controls or rats given estrogen on day 5. There was no decidual reaction observed irrespective of the position
of the blastocyst within the lumen (figs.
20-22). None of the blastocysts was elongated and all retained an oval shape; most
were smaller than those seen in control
animals (figs. 23-25).
The uteri of rats killed on day 6, but
given dienestrol on day 5 were in general
intermediate in appearance between those
of controls and of rats treated on day 4 but
more closely resembling the former. There
was some convolution of the uterine lumen
(fig. 26), but this was much less marked
than in the rats given estrogen on day 4.
In general the position of the blastocysts
within the lumen was normal (fig. 26).
Of 27 blastocysts observed, only two had
the embryonic pole directed towards the
anti-mesometrial surface (fig. 27), and the
orientation of a further four could not be
precisely determined. Some blastocysts
were elongated, but despite this were
439
smaller than those in control rats (fig. 28).
Although the epithelium was pressed close
against the blastocyst often no implantation chamber was formed (fig. 29). Indeed
in some uteri the lumen was quite wide.
The epithelium at the site of blastocyst apposition showed increased height similar
to that in control rats but the nuclei of
these cells were not on the basal membrane
and sub-nuclear vacuoles were present
(figs. 28-30). In some cases the epithelium was tightly pressed against the blastocyst (figs. 28,29), but despite this it seemed
that the blastocysts were not attached to
the epithelium at either the embryonic or
the abembryonic pole (figs. 28-30). The
epithelial cells had distinct boundaries,
while in the control rats the outline was
indistinct in the region of the abembryonic
pole of the blastocyst (figs. 16-19). There
was a small decidual reaction at the antimesometrial end of the lumen around the
position of the blastocyst (fig. 26).
DISCUSSION
Edgren and Shipley ('61) suggested that
estrone prevented pregnancy, when given
on days 1 or 2, by causing retention of eggs
in the oviduct, and after day 3 by interfering with the uterine reaction, They were,
however, unable to explain the effectiveness of estrone given on day 3, at a time
when the eggs were entering the uteri.
Saunders ('65) in his study with estrone
did not indicate its mode of action. Davis
('63) stated that norethynodrel given to
rats early in pregnancy could produce interference with egg transport and at the
time of implantation prevention of decidual
formation. These results were attributed
to the inherent estrogenicity of the compound.
The present experiments with dienestrol
have also provided evidence for two mechanisms of action operating either independently or concurrently depending on
the day of treatment. In table 5 the results
of Edgren and Shipley ('61) and Saunders
('65) have been converted to MEDs in
mg/kg and compared with those for the
present experiments. The results for estrone are not significantly Werent between experiments, and dienestrol although
given orally also produces similar figures.
Both Edgren and Shipley ('61) and Saunders
440
M. J. K. HARPER
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(’65) reported that the day of maximal
sensitivity was day 3. From Saunders’ results (Zoc. cit.) it is also clear that the
slope of the dose-response line for animals
treated on day 4 was very flat (6.79), although not significantly different from
those for other days of treatment. Emmens
and Finn (’6%) observed a flat dose-response line for percentage of rats pregnant
after either local or subcutaneous injections of estracliol on day 4 of pregnancy.
In the present experiments with dienestrol
the dose-response line on day 4 was also
flat (slope = 6.32). Dienestrol did, however, differ from estrone since although it
was most effective on day 3, the decrease
in MED from day 1 and day 2 to day 3 was
not as great as for estrone. In addition
dienestrol was apparently much less eff ective than estrone when given on day 5 although this may have been due to delaying
treatment until late on that day (1700
hours).
The results of these experiments support the notion that different modes of action of estrogen could account for the
changes in thie dose required to interrupt
pregnancy. However the results presented
in table 2 do not support the contention of
Edgren and Shipley (’61) concerning
“tubal-locking” of eggs. It is clear that
treatment on days 1, 2 or 3 causes premature entry of the eggs into the uterus,
followed by expulsion through the cervix.
Greenwald (‘61,’67) has reported that injection of pregnant rats with estradiol
cyclopentylpropionate on the morning that
sperm were found in the vaginal smear increased tubal and uterine mobility with
consequent expulsion of eggs from the
genital tract. “Locking” of eggs in the
oviduct was not observed by Greenwald
(’67), and this is in accord with the present findings.
The flat dose-response line observed after
treatment on day 4 can perhaps be explained in the light of the findings presented in table 3. Administration of the
minimal 100% effective dose of dienestrol
on day 4 caused expulsion of some, but
not all, eggs from the uterus by day 5. By
day 6 the percentage of eggs recovered
from the uterus (51%) was not significantly different from the controls (47% ),
and so the failure of these eggs remaining
M E C H A N I S M O F E S T R O G E N ACTION O N EARLY P R E G N A N C Y
in the uterus to form implantation swellings must be due to an effect of estrogen
on the uterus, since the development of
the blastocysts appeared normal. Prevention of pregnancy after estrogen treatment
on day 5 must be solely due to the uterine
effect, since expulsion of eggs from the
uterus did not occur. Thus it is only on
day 4 that both mechanisms (expulsion of
eggs and an effect on the uterus) play a
role in prevention of pregnancy.
The nature of this uterine effect was
initially obscure since although failure of
the gross deciduomal response to trauma
occurs, it does so at a lower dose than that
at which implantation fails (compare
tables 1, 2). Furthermore the effect of
excess estrogen on the pontamine blue reaction was not consistent. If dienestrol
was given on day 4, a reduction in the
number of rats showing a general blue reaction of the uterus on day 6 was seen,
and uniformly negative results were obtained on days 7 and 8. Dosing on day 5
had very different effects since fairly normal uterine blue reactions were seen, but
delayed by one day compared to control
rats. The blastocysts recovered from the
uteri of treated rats looked no different
from those obtained from control rats after
the time of initial attachment. The blebbing from the blastocyst surface may represent evidence of initial attachment and
the subsequent disruption of this attachment by the flushing procedure. However
it must be noted that this phenomenon was
seen just as commonly in blastocysts recovered on day 6 from animals treated on
day 4 as those from controls or animals
treated on day 5 and yet only one such rat
showed a normal reaction to pontamine
blue on the same day. A photomicrograph
of a blastocyst showing a somewhat similar appearance has been described by
Alloiteau and Psychoyos ('66) as a 'blastocyste Q trophoblaste bourgeonnant," although in this case the blastocysts had
developed in the ligated oviducts of hypophysectomized rats.
A study of the histological sections of
the uteri from control and estrogen treated
rats killed on day 6 clarified this uterine
effect. Estrogen treatment on day 4 disrupted both the positioning of the blastocyst in the lumen and its orientation rela-
44 1
tive to the mesometrial-anti-mesometrial
axis. The lumen was extremely convoluted
and the epithelium increased in height
over the whole luminal surface and not
just at the site of blastocyst apposition.
There was no indication of any reaction
between the blastocyst and the epithelium
or underlying tissue (i.e., no decidual
reaction and no attachment to the epithelium). The epithelium also showed pseudostratification which Psychoyos ('66) observed in mated rats ovariectomized on day
4, given no steroid treatment and killed on
day 9. These findings clearly explain the
lack both of a pontamine blue reaction in
most rats and of implantation swellings.
They also suggest that the cellular protrusion observed in the blastocysts flushed
from the uterus was not necessarily any
indication of attachment to the epithelium.
The effect produced by administration of
estrogen on day 5 was considerably less pronounced than when it was given on day 4.
Obviously some effect on orientation of the
blastocysts had occurred and there was
some convolution of the lumen. These factors alone, however, would be unlikely to
lead to a total failure of implantation. Furthermore on day 6 there was some decidual
reaction around the area of blastocyst apposition, although less than in controls.
In the rat the pontamine blue reaction normally precedes the decidual changes
(Psychoyos, '60, '61, '67). It is somewhat
surprising therefore that no blue reaction
was observed in the estrogen-treated rats
until day 7, i.e., 24 hours after the appearance of the decidual changes. However
study of the histological sections of the
day 6 uteri suggested that no attachment
at either the embryonic pole or the abembryonic pole had commenced. Attachment
to the epithelium on day 5 of pregnancy
first occurs at the embryonic pole and at
the same time (1200-1600 hours) the blue
reaction first becomes visible (Psychoyos,
'66). Perhaps attachment in these estrogen-treated rats was delayed until day 7,
and the appearance of the blue reaction on
that day indicated its occurrence. Such
decidual reaction as there was on day 6
was apparently not maintained until day
9, since it was absent from the uteri of the
pseudopregnant rats given the same dose
of estrogen at the time of uterine trauma
442
M. J. K. HARPER
on day 5. The blue reaction on day 7 (in
the pregnant rats) suggests that the decidual reactions was, however, maintained
until that day. The reversed order of appearance of the blue coloration and the decidual reaction shows that the normal course
of events had been grossly disturbed and
this could have been sufficient to prevent
the blastocyst (when and if it attached)
from forming an implantation swelling.
An interesting observation was made in
these rats given excess estrogen on day 5
and killed on day 6. Sub-nuclear vacuoles
were still present in the epithelial cells. In
the normal pregnant rat such vacuoles disappear on day 5 as a result of the estrogen
intervention which induces implantation
(Psychoyos, ’66). Psychoyos (’66) has
surmised that these vacuoles are the site
of the lipid droplets described by Alden
(’47), and suggests that their migration
upward through the cytoplasm is a n index of estrogen intervention. This was confirmed by an electron-microscopic study of
the uteri of rats with delayed implantation
and of similar rats 18 to 21 hours after a
physiological dose of estrogen (Potts and
Psychoyos, ’67). In the present experiments, however, excess estrogen has either
prevented the dispersion of this pre-formed
lipid or caused fresh formation.
Psychoyos (’67) has suggested that in
the early stages of the implantation reaction the blastocyst is compressed by the
swelling of the stroma and it is at this time
that the increase in capillary permeability
denoted by the blue reaction is first seen. In
rats given excessive doses of estrogen on
day 5 decidualization commences before
blastocyst attachment and the increased
capillary permeability. Despite this no
blastocyst attachment was seen in the absence of a blue reaction and decidualization. In addition blue patches did not appear until after blastocyst elongation. This
elongation is accompanied by the increase
in size of the blastodemic vesicle and
flattening of the ectodermal cells (Huber,
’15). Elongation, however, does not seem
to be due to stromal compression since in
the rats given estrogen on day 4 the blastocysts were often found in crypts tightly
pressed by the epithelium but not elongated.
Noyes and Dickmann (’60) have postulated that implantation will only occur
when the “ovum and the endometrium
have simultaneously reached specific stages
in their development by the time they come
in contact with each other.” The present
experiments do not entirely support this
hypothesis, since initial attachment not
only could occur later than normal (viz.,
blue patches on day 7 - table 4), but
could also occur in a uterus with minimal
ability to form decidua (table 2). However, it is clear that lack of adequate synchronization of blastocyst and endometrium is inimical to continued normal
development. The primary function, therefore, of the decidual reaction is to support
the continued growth of the blastocyst after
attachment rather than to facilitate such
attachment. Indeed blastocyst compression
by and attachment to the epithelium of
the crypt is the prerequisite for the pontamine blue reaction and for decidual induction in a normal pregnancy.
ACKNOWLEDGMENTS
I thank Mrs. Audrey Smith and Mrs. Ann
S. W. Elliott for technical assistance.
LITERATURE CITED
Alden, R. H. 1947 Implantation of the rat egg.
11. Alteration in osmiophilic epithelial lipids
of the rat uierus under normal and experimental conditions. Anat. Rec., 97: 1-13.
Alloiteau, J. J., and A. Psychoyos 1966 Y-a-t-il
pour l’oeuf de ratte deux faqons de perdre sa
zone pellucide? C. R. Acad. Sci., Paris, 262:
1561-1564.
Davis, B. K. 1963 Studies on the termination
of pregnancy with norethynodrel. J. Endocr.,
27: 99-106.
Dreisbach, R. H. 1959 The effects of steroid
sex hormones in pregnant rats. J. Endocr., 18:
271-277.
Edgren, R. A., and G. C. Shipley 1961 A quantitative study of the termination of pregnancy
in rats with estrone. Fert. Steril., 12: 178-181.
Emmens, C. W., and C . A. Finn 1962 Local
and parenteral action of oestrogens and antioestrogens on early pregnancy in rat and mouse.
J. Reprod. Fert., 3: 239-245.
Greenwald, G. S . 1961 The anti-fertility effects
in pregnant rats of a single injection of estradiol cyclopentylpropionate. Endocrinology, 69:
1068-1073.
1967 Species differences in egg transport in response to exogenous estrogen. Anat.
Rec., 157: 163-172.
Huber, G. C. 1915 The development of the albino rat ( M u s noruegicus albinus). I. From
the pronuclear stage to the mesoderm anlage;
MECHANISM O F ESTROGEN ACTION ON EARLY PREGNANCY
end of the first to the end of the ninth day. J.
Morph., 26: 247-358.
Litchfield, J. T., Jr., and F. Wilcoxon 1949 A
simplified method of evaluating dose effect experiments. J. Pharmac. Exp. Ther., 96: 99113.
Marston, J. H., and M. C. Chang 1964 The
fertilizable life of ova and their morphology
following delayed insemination in mature and
immature mice. J. Exp. Zool., 155: 237-251.
Noyes, R. W., and Z. Dickmann 1960 Relationship of ovular age to endometrial development.
J. Reprod. Fert., I: 186-196.
Parkes, A. S., and C. W. Bellerby 1926 Studies
on the internal secretions of the ovary. 11. The
effects of injection of the oestrus producing
hormone during pregnancy. J. Physiol. Lond.,
62: 145-155.
Potts, M., and A. Psychoyos 1967 Evolution de
l'ultrastructure des relations ovoendometriales
sous l'influence de l'oestrogkne, chez la ratte
e n retard experimental de nidation. C. R.
Acad. Sci., Paris, 264: 370-373.
Psychoyos, A. 1960 La reaction dbciduale est
prCckdCe de modifications prkcoces de la per-
443
mCabilit6 capillaire de l'uterus. C. R. SOC.Biol.,
154: 1384-1387.
1961 PermCabilite capillaire e t decidualisation ut6rine. C. R. Acad. Sci., Paris, 252:
1515-1517.
1966 Btude des relations de l'oeuf et
de l'endombtre a u cours du retard de la nidation ou des premieres phases du processus de
nidation chez la ratte. C. R. Acad. Sci., Paris,
263: 1755-1758.
1967 The hormonal interplay controlling egg-implantation in the rat. In: Advances
in Reproductive Physiology. Ed. by A. McLaren.
Logos Press, London, 2: 257-277.
Saunders, F. J. 1965 Effects o n the course of
pregnancy of norethynodrel with mestranol
(Enovid) administered to rats during early
pregnancy. Endocrinology, 77: 873-878.
Smith, M. G. 1926 On the interruption of pregnancy in the rat by the injection of ovarian
follicular extract. Bull. Johns Hopk. Hosp.,
39: 203-214.
Yanagimachi, R., and M. C. Chang 1961 Fertilizable life of golden hamster ova and their
morphological changes a t the time of losing
fertilizability. J. Exp. Zool., 148: 185-204.
PLATE 1
EXPLANATION OE’ FIGURES
All figures were photographed under a phase-contrast microscope in a
fresh condition and without compression. Magnification x 535.
2-6
Blastocysts recovered from control rats on day 6 of pregnancy.
2
Appearance of a normal five day blastocyst after loss of zona.
3-5
444
Blastocysts showing varying degrees of blebbing.
6
A n unusually large blastocyst.
7
A blastocyst recovered on day 6 from a rat given dienestrol (1 mg/kg
p.0.) on day 4 showing one cell protruding from the abembryonic
surface of the trophoblast.
MECHANISM OF ESTROGEN ACTION ON EARLY PREGNANCY
M. J. K. Harper
PLATE 1
445
PLATE 2
EXPLANATION OF FIGURES
Figures 8-13 were photographed under a phase-contrast microscope in
a fresh condition and without compression. Magnification x 535.
Figure 14 was photographed using bright fielc! illumination after h a tion with AFA and staining with hematoxylin and eosin. Magnification
X 104.
8-10
Blastocysts recovered on day 6 from rats given dienestrol (1mg/
kg p.0.) on day 4 showing varying degrees of blebbing. Compare
with blastocysts from control rats (figs. 3-5).
11-13
Blastocysts recovered on day 6 from rats given dienestrol (1 mg/
kg p.0.) on day 5 showing varying degrees of blebbing. Note the
smaller size compared to blastocysts in figures 8-10.
14 A cross section of the uterus of a control rat o n day 6 of pregnancy
showing a narrow straight lumen, a n elongated blastocyst and an
early decidual reaction ( d ) a t the anti-mesometrial ( a m ) end of
the lumen.
446
MECHANISM OF ESTROGEN ACTION ON EARLY PREGNANCY
M. J. K. Harper
PLATE 2
447
PLATE 3
EXPLANATION OF FIGURES
All the figures on this plate were photographed using bright field illumination after fixation with AFA and staining with hematoxylin and eosin.
All photographs were taken parallel to the mesometrial-anti-mesometrial
axis and are mounted so that the mesometrial edge of the uterus is on the
left side of the plate and the anti-mesometrial one o n the right. Magnification, figures 15, 20-22 x 104 and figures 16-19 x 415.
15-19
Sections of uteri of control rats killed on day 6 of pregnancy.
15 Transverse section showing a wider lumen than in figure 14 due
to a shrinkage arteract. Note the elongated blastocyst and the
decidual reaction ( d ) .
16-19
Blastocysts lying in crypts in the epithelium. The shape of the
crypt shows that blastocyst and epithelium had been closely apposed before blastocyst shrinkage. The epithelium is significantly
increased in height around the abembryonic pole of the blastocyst
and the outline of the cell boundaries is indistinct indicating the
site of attachment ( a ) . Note that the nuclei of the epithelial
cells are on the basal membrane. All blastocysts are correctly
oriented and elongated.
20-22
Transverse sections of uteri from rats killed on day 6 but given
dienestrol (1 mg/kg p.0.) on day 4. The lumen is very convoluted
and the blastocysts are i n the center rather than at the anti-mesometrial end of the lumen. Note the complete absence of any decidual reaction. The blastocyst is correctly oriented in figure 20,
but not i n figures 21 and 22.
MECHANISM OF ESTROGEN ACTION ON EARLY PREGNANCY
M. J. K. Harper
PLATE 3
449
PLATE 4
EXPLANATION OF FIGURES
All figures were photographed using bright field illumination after fixation with AFA
and staining with hematoxylin and eosin. All photographs were taken parallel to the
mesometrial-anti-mesometrial axis and are mounted so that the mesometrial edge of
the uterus is on thc left side of the plate and the anti-mesometrial one on the right.
Magnification, figures 23-25 and 27-30 x 415 and figure 26 x 104.
23-24
Sections of uteri of rats killed o n day 6 and given dienestrol (1 mg/kg p.0.)
on day 4. Note the increased height of the epithelium over the whole luminal
surface, and not just at the abembryonic pole of the blastocyst. Compare with
controls (figs. 16-19) and rats given estrogen on day 5 (figs. 27-30). The
boundaries of the epithelial cells adjacent to the blastocyst surface are distinct,
denoting absence of attachment. There is pseudo-stratification (ps) of the
epithelium in figures 23 and 24. The nuclei of the epithelial cells are on the
basal membrane.
23 The embryonic pole of the blastocyst is directed towards the anti-mesometrial
edge of the uterus.
24 The embryonic pole is transverse to the mesometrial-anti-mesometrial axis.
25 This blastocyst is correctly oriented.
26-30
Sections of uteri of rats killed o n day 6 but given dienestrol (1 mg/kg p.0.) on
day 5.
26 Transverse section showing relatively narrow but more convoluted lumen than
in controls (figs. 14, 15). Some decidual reaction ( d ) around the blastocyst
crypt can be seen and the blastocyst is partially elongated.
27-30
Blastocysts lying in crypts in the epithelium. Some of the blastocysts are
partially elongated, but in figures 28 and 29 the crypts are quite narrow. The
blastocysts had been closely apposed to the epithelium (compare with controls
figs. 16-19) but in general the boundaries of the epithelial cells are distinct.
The epithelium is increased in height a t the anti-mesometrial surface. Note
that the nuclei of the epithelial cells are not on the basal membrane and there
are vacuoles ( v ) between the nuclei and the basal membrane. These denote
the presence of lipid droplets.
27 The embryonic pole of this blastocyst is directed towards the anti-mesometrial
surface.
28-30
450
These blastocysts are correctly oriented.
MECHANISM OF ESTROGEN ACTION ON EARLY PREGNANCY
M. J. K. Harper
PLATE 4
451
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