/. Embryo/, exp. Morph. Vol. 55, pp. 109-122, 1980
Printed in Great Britain © Company of Biologists Limited 1980
109
Evidence of prostaglandin involvement in
blastocyst implantation
By P. V. HOLMES 1 AND B. J. GORDASHKO 2
From the Division of Morphological Science, Faculty of Medicine,
The University of Calgary, Canada
SUMMARY
Delayed-implantation mice were used to test for local implantation induction effects of
prostaglandins E2 and F 2 a . Evidence of implantation was gathered by microdissection of
implantation sites and by scanning electron microscopy. Indomethacin was tested for the
ability to interfere with the mechanisms of estrogen-induced, normal implantation. Both
prostaglandins appear to have significant effect inducing implantation when applied locally
in the uterine lumen, PGE2 being more effective than PGF 2a . The indomethacin clearly has
a blocking effect on normal implantation. However, indomethacin appears only partially able
to prevent the morphological changes indicative of the trophoblast cell transformation.
INTRODUCTION
During delayed-implantation in the female mouse, diapausing blastocysts
with virtually no proliferative activity lie in the luminal milieu of a progesteronequiescent uterus. Estrogen molecules introduced into this system rapidly activate
the whole system, resulting in implantation and the beginning of pregnancy
(McLaren, 1971). The precise location of the activating mechanism for this
system, of course, must involve estrogen receptor molecules. Estrogen receptors
have been researched considerably in uterine tissue and evidence now exists
showing they are probably also present in pre-implantation blastocysts of mice
and rabbits (Holmes, 1976). Despite these findings, the estrogen mechanism for
blastocyst activation and implantation remains obscure.
In the blastocyst an estrogen stimulus rapidly increases DNA, RNA and
protein synthesis (Weitlauf & Greenwald, 1965; Prasad, Dass & Mohla, 1969;
Gulyas & Daniel, 1969; Sanyal & Meyer, 1970; Inoui, 1971; Holmes & Dickson,
1975) which, in turn, activate enzyme systems in the trophoblast cells (Christie,
1967; Wong & Dickson, 1969; Holmes & Dickson, 1973; Holmes & Bergstrom,
1976). These activated trophoblast cells alter their surface adhesion properties
(Holmes & Dickson, 1973; Nilsson, Lindqvist & Ronquist, 1975; Bloxham &
1
Author's address: Dr P. V. Holmes, Stangebergsvagen 7, S-421 68, Vastra Frolunda,
Sweden.
2
Author's address: Division of Morphological Science, Faculty of Medicine, The University of Calgary, Calgary, Alberta, Canada.
8
EMB
55
110
P. V. HOLMES AND B. J. GORDASHKO
Pugh, 1977) and their morphology (Wu & Meyer, 1974; Bergstrom & Nilsson,
1976).
In 1973, Lau, Saksena & Chang published evidence that indomethacin
blocked ovo-implantation in mice. They initially suggested that the blocked
function was in the oviducal transport of the early ova. However, later evidence
from their laboratory (Lau & Chang, 1975) showed that the indomethacin
specifically blocked the ovo-implantation induced by exogenous estrogen in
delayed-implantation mice, thus avoiding oviducal transport effects. In addition,
their findings (Lau et al. 1973) demonstrated that prostaglandins E2 and F 2a
could reverse the anti-implantation effect of the indomethacin.
The above work introduces the possibility that a prostaglandin may lie on the
pathway for estrogen activation of the blastocyst implantation system. Furthermore, although it remains unknown whether the estrogen stimulates the blastocyst or the endometrium, it is likely that local hormone functions are also
involved.
The present work includes two classical methods to test whether a local
prostaglandin could be involved in the estrogen-induced implantation of
blastocysts. The first method replaces the estrogen stimulus with a prostaglandin
stimulus which is applied locally in the uterine lumen. The second attempts to
block normal implantation with indomethacin, an inhibitor of prostaglandin
synthesis. This should show whether prostaglandin production is necessary for
induction of implantation. The success of these two methods is controlled
morphologically using the scanning electron microscope (Bergstrom, 1972;
Holmes & Bergstrom, 1976) and physiologically by confirming and recording the
implantation successes.
MATERIALS AND METHODS
Swiss Webster albino mice were used from a random-bred colony maintained
at 22 °C with lighting controlled to provide a 10 h night centred on midnight.
Mouse food from Tecklad Inc. of Monmouth, Illinois and drinking water were
provided ad libitum. Surgical procedures were conducted under intraperitoneal
sodium pentobarbital anesthesia and bilateral ovariectomies were done on
day 3 of gestation, a vaginal plug having been found on day 1. At ovariectomy
0-5 mg of long-acting progesterone (Depo Provera, Upjohn, medroxyprogesterone acetate 50mg/ml) was administered subcutaneously producing
experimental diapause (Dickson, 1969).
Table 1 illustrates the treatment groups, the first group containing unoperated, untreated mice and representing the normal implantation controls.
The second group of ovariectomized, progesterone-treated mice are the delayedimplantation controls. An exogenous estrogen stimulus for implantation
(estradiol benzoate 005 jug subcut. in corn oil) was given to half of these mice
on day 8, the other half receiving subcutaneous corn oil only. Group-3 mice were
111
Prostaglandins induce implantation
Table 1. Mouse treatment groups
Gestation
day
1
Normal
control
mice
Vag. plug
Delayedimplantation
control mice
Vag. plug
2
Ovariectomy +
progesterone
3
4
5
8
Implantation
Estrogen or
vehicle t
Autopsy
Indomethacintreated*
normal mice
Prostaglandintreated
delayed mice
Vag. plug
(indomethacin)
or vehicle
(indomethacin)
or vehicle
(indomethacin)
or vehicle
(indomethacin)
or vehicle
Vag. plug
Ovariectomy +
progesterone
PGE2 or PGF 2a
or vehicle
Autopsy
Autopsy
Autopsy
14
* Administered in different combinations of days 2, 3, 4 and 5. See Table 2.
| Vehicle: corn oil with estrogen control mice, peanut oil with indomethacin controls,
and buffered saline with prostaglandin controls.
normal mice comparable to group 1 except that indomethacin was administered
subcutaneously in peanut oil according to the time and dosage schedules in
Table 2.
Group-4 mice were delayed-implantation mice as in the group-2 controls
except that they were treated with a prostaglandin on day 8. Prostaglandins E2
and F 2 a dissolved in phosphate-buffered saline at pH 7 were given by microsyringe as intra-uterine instillations, 6 ju\ per uterine horn. The dosages of
PGE 2 and PGF 2a can be seen in Tables 3 and 4, respectively.
Just prior to instillation of experimental or control fluids into the uterine
lumen, a cotton ligature was placed around the upper cervix, avoiding major
blood vessels in that region. This procedure minimized the escape of fluids and
the flushing of blastocysts from the uterus.
Numerous pilot studies were conducted initially to determine suitable doses
and times of administration for the indomethacin and the prostaglandins. PGE 2
(U-12062, Lot no. 5-PRC-3001A) and PGF 2a (U-14583E, lot no. 983BX,
trimethamine salt) were provided by The Upjohn Company, Toronto. The
experimental groups of mice in Table 1 included their own within-group controls
for the solvent vehicles of the hormones and blocker when it was deemed
necessary.
On day 14 of gestation all mice were autopsied with careful examination by
micro-dissection for uterine implantation sites. All unimplanted uterine horns
and unimplanted sections of horns were flushed with 2-5 % glutaraldehyde in
0-1 M Soerensen's buffer at pH 7-4. Any blastocysts collected were rinsed in
8-2
112
P. V. HOLMES AND B. J. GORDASHKO
Fig. 1. Delayed-implantation mouse blastocyst without endogenous or exogenous
estrogen stimulation. Since this non-activated blastocyst is held tightly in the
uterine lumen, it has an unexpanded form, a ridge encircling its long axis and
surface irregularities in the trophoblast cells. The micrograph to the right at higher
magnification shows that borders between the large polygonal trophoblast cells are
barely visible, while their surfaces are imprinted by the smaller uterine epithelium
cells, x 950 and x 9500.
Fig. 2. Delayed-implantation mouse blastocyst after in vivo activation by exogenous
estrogen given 26 h prior to collection. This blastocyst is larger than the unactivated
blastocyst in Fig. 1 -140/*m compared with 90 /*m from the embryonic to the
abembryonic poles. Individual trophoblast cells are expanded and bulging with
distinct, raised cell borders. The imprints of uterine epithelium cells are no longer
present. Also, bump-like formations are already evident on numerous cells which
seem to develop into globular protrusions at later stages of implantation, x 570
and x 1400.
Prostaglandins
induce
113
implantation
Table 2. Indomethacin treatment groups
Indomethacin
dosage/mouse
Og in
0-1 ml oil)
Day of
treatment
No. mice
treated
Oil controls
All groups
31
4
11
7
3
15
5
9
225
225
225
75
150
75
150
Mice with
implantations
1
2
2
12
12
3 4
3 4
3
3
3
5
5
4
4
4
6
6
Mice with
blastocysts
>
No.
29
3
3
3
1
1
4
4
/o
94
75
27
43
33
7
80
44
No.
0
0
3
1
0
7
0
0
/o
0
0
27
14
0
47
0
0
Line 1 includes the controls from all treatment groups in the table.
When the number of mice treated is greater than the mice with implantations plus those
with blastocysts, then the difference is the number of non-pregnant mice plus the mortalities
due to treatment.
buffer, post-fixed in buffered 2 % OsO4 for 30 min, rinsed in three baths of
redistilled water, and prepared by freeze-drying for examination in a Cambridge
Stereoscan electron microscope at 20 kV. Observations with the scanning EM
were made using a double-blind technique and the blastocysts were scored
according to characteristics described and utilized previously (Bergstrom, 1972;
Holmes & Bergstrom, 1976).
RESULTS
Group 1. Normal control mice. All of the eight mice used possessed implantation sites at autopsy on day 14 and no blastocysts could be collected for
scanning EM observations. A total of 37 sites, 4-5 mm diameter, were found.
The representative sites taken from each mouse appeared normal on microscopic
dissection.
Group 2. Delayed-implantation control mice. Six mice received an estrogen
stimulus on day 8 and, when autopsied on day 14, all mice were implanted.
A total of 34 implantation sites were found giving an average of 5-7 sites per
mouse. These sites were all approximately 3 mm diameter and apparently
normal. A second group of six received control corn-oil injections on day 8 and
these mice completely lacked implantations at autopsy. Instead 27 blastocysts
were collected and confirmed by light microscopy and scanning EM to be in a
diapausing, unstimulated condition, comparable to those observed by Dickson
(1967, 1969) and Bergstrom (1972). From Fig. 1 the diapausing blastocyst can
be seen to have a rough surface formed by multiple imprints from the smaller
uterine epithelium cells. The crater-like imprints present are due to bulbous
protrusions from epithelium cells. The cell border outlines of the much larger
trophoblast cells are indistinct. Furthermore, diapausing blastocysts usually
114
P. V. HOLMES AND B. J. GORDASHKO
Fig. 3. Estrogen-stimulated mouse blastocyst treated with indomethacin, an
inhibitor of prostaglandin synthesis. Trophoblast cells are expanded but not
bulging and globular protrusions are evident on some cells of the abembryonic
pole. The borders between trophoblast cells are hidden in intercellular troughs
instead of being distinct and raised as in normal activation, x 1450 and x 4800.
Prostaglandins induce implantation
115
Table 3. Prostaglandin E2 treatment on day 8 of gestation
Prostaglandin
Mice with
dosage/mouse
implantations
No. mice rOg in
No.
6/tl saline)
treated
/o
Saline controls
10
20
30
50
28
12
10
11
20
0
11
8
8
12
0
92
80
73
60
Mice with
blastocysts
A
No.
%
24
1
2
5
9
86
8
20
45
45
Seventy-seven implantation sites were found in the 39 implanted mice, giving an implantation rate of 2-0 sites/mouse.
Line 1 includes the controls from all treatment groups in the table.
When the number of mice treated is greater than the mice with implantations plus those
with blastocysts, then the difference is the number of non-pregnant mice plus the mortalities
due to treatment. When the number of mice treated is less, then at least one mouse had
implantation sites and blastocysts.
have a ridge encircling their longest circumference due to the grasp effect by the
uterus and the epithelial cells. This encircling ridge is very obvious in Fig. 1.
Fig. 2 is provided for comparative purposes. It is a scanning micrograph of a
completely activated blastocyst collected from a delayed-implantation mouse
that received exogenous estrogen 26 h prior to collection. Note the expanded,
polygonal trophoblast cells with convex surfaces, the distinct raised cell borders
and the absence of imprints from epithelial cells. An interesting detail are the
raised, bump-like formations evident on many cells. These appear to become
more numerous and more globular as the blastocyst approaches the time of
adhesion to the epithelium.
Group 3. Indomethacin-treated normal mice. Table 2 exhibits the treatment
schedules and the resultant implantation capabilities. The most successful
schedule for inhibiting implantation was 150 /ig indomethacin given on days 1,
2, 3 and 4, only one of the 15 mice having implantations. However, the mortality
rate was also high in this group, three mice died and blastocysts could be
flushed from only 7 of the 15 survivors. Fig. 3 illustrates the surface morphology
of an indomethacin-inhibited blastocyst and, although it appears partially
activated when compared to Figs. 1 and 2, the cell borders are not distinct or
protrusive. The trophoblast cells are rather flat and they are covered by small
microvilli. The cell surface bumps on this blastocyst appear to be protruding
globules possibly involved in a secretion process. All of the indomethacintreated blastocysts gave the impression they had been treated by an impeded
estrogen (Bergstrom, personal communication), morphological features of
trophoblast activation being present but without the implantation success.
Group 4. Delayed-implantation mice treated with prostaglandins. Tables 3
and 4 illustrate the implantation-inducing effects of PGE 2 and PGF 2a ,
116
P. V. HOLMES AND B. J. GORDASHKO
Table 4. Prostaglandin F2a. treatment on day 8 of gestation
Prostaglandin
dosage/mouse
Og in 6 /A
saline)
Mice with
implantations
No. mice
treated
Saline controls
5
10
20
30
24
8
7
9
8
A
,
Mice with
blastocysts
*
A
,
*
No.
%
No.
%
0
4
5
4
3
0
50
71
44
35
19
2
4
6
5
79
25
57
67
63
Twenty-six implantation sites were found in the 16 implanted mice, giving an implantation
rate of 1-6 sites/mouse.
Line 1 includes the controls from all treatment groups in the table.
When the number of mice treated is greater than the mice with implantations plus those
with blastocysts, then the difference is the number of non-pregnant mice plus the mortalities
due to treatment.
respectively. The most effective functioning prostaglandin was PGE 2 at the 10/tg/
6 [A dose level. This level gave no mortalities, 11 of 12 mice became pregnant and
activated blastocysts were flushed from the twelfth. All implantation sites were
2-3 mm diameter and appeared normal under microscopic dissection. Fig. 4
shows a well-expanded PGE2-activated blastocyst having distinct trophoblast
cells with raised cell junctions and low almost non-existent microvilli. All
trophoblast cells appeared activated, contrary to the indomethacin blastocysts.
In addition, globular protrusions were present on the outer surface of most of
the cells.
Fig. 5 exhibits the opposing poles of a partially expanded blastocyst after
treatment with PGF 2a . Both poles were illustrated here since the PGF 2a treatment showed itself to be rather slow or weak in transforming all trophoblast
cells for implantation. This is a typical half-transformed ovum, transformation
commencing with the abembryonic trophoblast cells as seen in the upper micrograph of Fig. 5 and progressing over the embryonic pole, seen in the lower
micrograph still in the untransformed condition. Although the abembryonic cells
are in the activation process and no longer bear imprints of uterine epithelial
cells, they are not fully expanded and are just beginning to develop the distinct,
FIGURE 4
Fig. 4. Delayed-implantation mouse blastocyst after treatment with intrauterine
instilled PGE2. These two micrographs exhibit a large expanded blastocyst with
characteristics of normal estrogen activation. Individually distinct polygonal
trophoblast cells are delineated by raised cell borders and are beginning to bulge
outwardly, the shallow uterine epithelial cell imprints being barely visible. Globular
protrusions are developing on most cells. The lower micrograph illustrates the
abembryonic pole of the blastocyst where trophoblast cell transformation begins,
x 750 and x 1375.
Prostaglandins induce implantation
117
118
P. V. HOLMES AND N. J. GORDASHKO
Prostaglandins induce implantation
119
raised cell borders. The trophoblast cells of the embryonic pole are not individually recognizable yet since their activation and expansion has not begun
and they remain masked by the extensive uterine epithelial imprints. No large
globular protrusions were seen on PGF2a-induced blastocysts.
In the 39 PGE2-treated mice with implantations in column 3 of Table 3,
77 implantation sites were found giving an implantation rate of 2-0 sites per
mouse. The 16 PGF2a-treated mice with implantations, in column 3 of Table 4,
exhibited 26 implantation sites giving an implantation rate of 1-6 sites per mouse.
These rates can be compared to the six estrogen-treated mice exhibiting 34
implantation sites and a rate of 5-7 sites per mouse. The rates above are calculated from all treated mice in Tables 3 and 4 without heed to dosages. Therefore
the rate figures do not reflect the best rate obtained from the most ideal PGdosage used.
If one compares the visual micrographic results with the implantation effects
summarized in Tables 3 and 4, implantation-induction by PGF 2a is not as
clear-cut as with PGE 2 . This could be attributed to a slower induction rate and
weaker pharmacological effect with PGF 2a in this particular biological system.
DISCUSSION
The findings in the present study provide good evidence that a prostaglandin,
in particular PGE 2 , is involved in the activating function of estrogen for
successful pregnancy. Implantation occurring in intact mice with their own
endogenous, activating surge of estrogen on day 4 (McCormack & Greenwald,
1974) was successfully blocked by indomethacin as seen in the present findings
and those of Lau et al. (1973). Implantation occurring in delayed-implantation
mice receiving an exogenous estrogen stimulus was also successfully blocked by
Lau & Chang (1975) and Saksena, Lau & Chang (1976) using indomethacin. The
present morphological findings illustrate that both PGE 2 and PGF 2a stimulate
changes in the trophoblast cells of the blastocyst, and are able to induce blastocysts to implant in the uterus. Furthermore, blastocysts from indomethacinblocked implantation in normal intact mice were impeded in their morphological
activation. This impediment in the complete transformation of the trophoblast
FIGURE 5
Fig. 5. Delayed-implantation mouse blastocyst after intrauterine treatment with
PGF 2a . The PGF2a-stimulation resulted in half-transformed, partially expanded
blastocysts. Such a blastocyst is exhibited here, abembryonic pole above and
embryonic pole below. Flat, partially activated trophoblast cells with cell borders
hidden in troughs encompass the abembryonic pole. The only globular protrusion
seen is exhibited here on an incompletely expanded abembryonic trophoblast cell.
The embryonic pole below has the typical characteristics of a diapausing blastocyst; remains of an encircling ridge, uterine epithelial cell imprints covering a
rough surface, and indistinct delineation of trophoblast cells, x 3000 and x 3000.
120
P. V. HOLMES AND B. J. GORDASHKO
cells, apparent in the scanning electron micrographs, can probably be attributed
to insufficient prostaglandin production.
The hypothesis that a prostaglandin molecule is in the pathway for estrogenactivation of the trophoblast-endometrium system is strengthened by research
demonstrating estrogen-stimulated prostaglandin production in the uterus,
particularly in decidual tissue (Barcikowski, Carlson, Wilson & McCracken,
1974; Anteby, Bauminger, Zor & Linder, 1975; Castracane & Jordan, 1975;
Ham, Cirillo, Zanetti & Kuehl, 1975; Kuehl, Zanetti, Cirillo & Ham, 1975).
Also, the trophoblast cell activation and implantation seen in the present
findings are supported by work of Williams & Downing (1977). They incubated
the microsomal fraction of rat decidual tissue and, using GLC, mass spectrometry and TLC techniques, were able to show the major tissue product to be
PGE 2 . In the present work, however, PGF 2a also had some activating and
implanting effect although less than PGE 2 .
Burstein, Gagnon, Hunter & Maudsley (1976) have demonstrated that PGE 2
stimulates cyclic AMP production in cultured epithelial cells, while PGF l a and
PGF 2a have only a very slight effect. Furthermore, Kuehl, Cirillo, Ham &
Humes (1973) found that, although all prostaglandins stimulate cyclic AMP
synthesis in intact tissues, E-prostaglandins were far superior to A and F types
in their potency. These PGE 2 effects on cyclic AMP could be linked with the
findings of Holmes & Bergstrom (1973,1975,1976) and Webb (1975). They were
able to positively induce implantation by stimulating delayed-implantation
mice with cyclic AMP. Thus, a relationship between PGE 2 and cyclic AMP is
implied. The possibilities suggested here are that the estrogen-activating stimulus
functions via a combined PGE2-cyclic AMP mechanism or that the stimulus
requires both separate prostaglandin and cyclic nucleotide pathways to complete
normal implantation. Since E-prostaglandins are known to stimulate cyclic
AMP formation in a dose-related manner (Wolf & Shulman, 1969; Bourne,
Lichtenstein & Melmon, 1972), the above possibilities could be tested for the
blastocyst-implantation system using embryo culture and labelling techniques.
Batta & Martini (1975) demonstrated in intact rats that intra-uterine injections of PGE 2 prior to implantation reduced the number of surviving implantations sites. However, since the surviving implantations were normal and produced
viable fetuses, the possibility exists that the hormone injections could have
expelled some ova from the uteri. This was not possible in the present work
due to the use of cervical ligatures. Furthermore, the prostaglandins injected by
Batta & Martini would have been additional to those already stimulated by
endogenous estrogen, making the uterine luminal environment non-physiological.
Related work also has been done in hamsters, work that supports the findings
here. Evans & Kennedy (1977) determined uterine tissue concentrations of
prostaglandins E and used the blocking effects of indomethacin to establish a
role for prostaglandins in decidualization and implantation. Additionally, these
Prostaglandins induce implantation
121
workers recently showed (Evans & Kennedy, 1978) that the two inhibitors of
prostaglandin synthesis, indomethacin and meclofenamic acid, reduced uterine
PG concentrations and inhibited the increase in uterine vascular permeability,
an important characteristic of the implantation process.
This research was supported by a grant from the Medical Research Council of Canada.
We are grateful to Dr E. L. Masson, M.D., of The Upjohn Company of Canada for the
generous supply of prostaglandins and we thank Bibi Karlsson for valuable assistance in
preparation of the manuscript.
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{Received 15 February 1979, revised 24 September 1979)