/ . Embryol. exp. Morph. Vol. 34, 2, pp. 299-310, 1975
Printed in Great Britain
299
The hormonal control and morphology
of blastocyst invasion in the mouse uterus
in vitro
By P. S. GRANT, 1 1. LJUNGKVIST 1 AND O. NILSSON 1
From the Reproduction Research Unit, Biomedical Center, Uppsala
SUMMARY
When mature and immature uteri from ovariectomized mice were cultured in chemically
defined media, blastocyst invasion occurred in the presence of progesterone but not in media
containing only oestradiol.
The invaded stromal tissue did not decidualize unless the uteri were taken from mice
pretreated with progesterone and oestradiol. Fibrillar material was, however, concentrated
in the stromal tissue adjacent to the invasive trophoblast.
Neither progesterone nor oestradiol, had any ultrastructural effect on the endometrium
of cultured uteri from ovariectomized mice, other than closure of uterine lumina in the
presence of progesterone.
Embryogenesis became abnormal, probably owing to the failure of formation of a suitable
implantation chamber.
INTRODUCTION
The process of egg implantation in the mouse is characterized in vivo by
blastocyst invasion of the endometrium, the transformation and growth of the
stromal cells into decidual tissue, and rapid embryonic development (Snell &
Stevens, 1966). These events are initiated and sustained by the combined
action of progesterone and oestrogen (Yoshinaga & Adams, 1966).
Blastocyst invasion of mouse uteri maintained in vitro is also subject to
hormonal control, but only progesterone is required (Grant, 1973a). Under
these conditions the stromal tissue does not decidualize and embryogenesis
becomes abnormal during egg-cylinder formation. The culture system used,
however, contained serum of unknown hormonal composition and the uteri
were from intact immature mice.
The present experiments were designed to answer the following questions:
(1) will progesterone also permit blastocyst invasion in uteri from ovariectomized mice cultured in chemically denned media? (2) will mature uteri allow
decidualization and more normal embryogenesis? (3) is the ultrastructure
of trophoblastic invasion in vitro comparable to that in vivo ?
1
Authors' address: Reproduction Research Unit, Box 571, Biomedical Center, S-751
23 Uppsala, Sweden.
300
P. S. GRANT, I. LJUNGKVIST AND O. NILSSON
MATERIALS AND METHODS
In vitro experiments
Uterine horns from NMRI mice were cultured using methods previously
described (Grant, 1973 a, b).
To test if progesterone permitted invasion, donor blastocysts were transferred
into the lumina of the uterine horns (3-8 per horn). Blastocysts were flushed on
the 5th or 6th day of pregnancy from mice ovariectomized at 2-5 days post
coitum (p.c.) and immediately given 1 -0 mg of medroxyprogesterone acetate
(Depoprovera, Upjohn). They were manipulated in phosphate-buffered saline
containing 2 % ovariectomized mouse serum.
The uteri were from adult or immature mice (ovariectomized at least 7 days
previously) or from intact immature mice. Furthermore, to examine the
influence of blastocyst manipulation, uteri containing native blastocysts from
adult mice were cultured. These mice were also ovariectomized 2-5 days p.c.
and their uteri were cultured 4-8 days later.
The culture media used for all experiments were Trowell's T-8 medium or
Eagle's Minimum Essential Medium (Glasgow modification) with 100 i.u. of
penicillin G per ml. The media were used unmodified (group 1), or with the
addition of progesterone (group 2), oestradiol (group 3), or progesterone and
oestradiol (group 4). Progesterone and oestradiol (both Sigma) were first
dissolved in ethanol and added to the media as 40 % aqueous solutions to give
final concentrations of 2-5 and 0-05 (or 0-006) /ig per ml, respectively.
Hormonally-induced ultrastructural changes in endometrial cells were also
sought in specific experiments with uteri from ovariectomized mature mice.
Horns from three mice were cultured in each of the treatment media, using the
higher level of oestradiol. Uteri from two mice were cultured in media containing 100 fig per ml of oestradiol.
To test if decidualization and more normal embryogenesis would accompany
invasion in mature uteri (here defined as uteri capable of supporting normal
embryonic development invivo), superovulated immature mice or adult pregnant
mice were ovariectomized as before and then pre-treated with progesterone
(1-0 mg, Leo; in 0-04 ml peanut oil s.c.) and oestradiol (0-05/*g, Sigma; in
0-1 ml peanut oil s.c.) 16-25 h before culture of their uteri, i.e. 7-11 days p.c.
Ten minutes before their death, these mice were injected intravenously with
0-15 ml of 1-0% Pontamine Sky Blue (Gurr) in 0-9% NaCl, to identify the
onset of decidualization in vivo.
Cultures were gassed with 27-95 % O2 in an atmosphere containing 5 % CO2
with the remainder N 2 . Within this range, the O2 concentration used was
approximately proportional to the diameter of the horns to be cultured.
Cultures were maintained at 35 °C for 2-3 days and then fixed by immersion in
2-5% glutaraldehyde in Sorensen's buffer (pH 7-3) while still pinned on the
culture grids. Two of the three horns in each treatment group cultured without
Blastocyst
invasion in mouse uterus in vitro
301
eggs were fixed by careful injection of the fixative through their lumina before
immersion.
For light microscopy (LM) after fixation all horns were dehydrated in
ethanol and embedded for longitudinal sectioning in methacrylate plastic (Ju.
Porter). For electron microscopy (EM) 3-4 mm long pieces of uteri were postfixed in osmium tetroxide and embedded in Epon 812.
Longitudinal sections were cut with an LKB-Pyramitome (3 jum for LM
and of variable thickness if an embryo was used for EM). Ultrathin sections
were cut with an LKB-Ultrotome, floated on to Formvar-covered grids and
stained with uranyl acetate and lead citrate, before examination in a JEOL
100 B electron microscope. Sections for LM were stained with haematoxylin
(Erhlich's) and eosin (0-5% alcoholic). Notes were made on tissue survival
after culture, and embryos with 33 % or more of their cells showing signs of
death were classified as dead.
In vivo experiments
To compare the response of uteri from immature mice in vitro with their
response in vivo, the hormonal requirements for egg implantation and the
adequacy of immature uteri for embryonic development were examined in
vivo. Donor blastocysts (identical to those used in the experiments in vitro)
were transferred to the uteri of mice aged around 3 weeks (10-14 g body weight)
during ether anesthesia. The mice were ovariectomized immediately after
transfer. Results are given in Table 2.
There were four treatment groups. Mice were injected daily subcutaneously
with 0-1 ml of peanut oil, alone (group 1) or containing 1-0 mg progesterone
(group 2), or 0-05 fig oestradiol (group 3) or 1-0 mg progesterone and 0-05 /^g
oestradiol as separate injections (group 4). Two or three days after ovariectomy,
mice were anesthetized with pentobarbitone sodium and their uteri fixed either
by heart injection with 2-5% glutaraldehyde in Sorensen's buffer or by
immersion after instillation of the fixative intraperitoneally. The uteri were
then prepared for light microscopy in the same way as were the cultured uteri.
Discrete data were analysed with contingency tables. Three orthogonal comparisons were used with 2 x 2 tables to examine the effects of the hormones
on embryonic development. These were (1) progesterone-treated groups
versus non-progesterone-treated groups, (2) progesterone-treated group versus
progesterone plus oestradiol-treated group and (3) the control group versus
the oestradiol-treated group.
RESULTS
In vitro experiments (Table 1)
Uterine horns from either adult or superovulated immature mice differed
from those of immature mice by having larger diameters and more complex
302
P. S. GRANT, I. LJUNGKVIST AND O. NILSSON
Table 1. The viability and development of eggs within the lumina of uterine horns
cultured in the presence of progesterone and oestradiol {numbers of uterine horns
and eggs)
Horns cultured ...
Native plus injected eggs*
Eggs found after culture
Eggs that were 'alive'
Trophoblast activity of 'live' eggs
With enlarged cells
That were invading the
epithelium
That were invading the stroma
Embryonic development of
'live' eggs|
That were in delay
With proximal endoderm
With distal endoderm
Beyond distal endoderm
Control
Progesterone
Oestradiol
Progesterone +
oestradiol
9
45
4
3
14
54
27
20
8
36
26
19
20
81
38
34
1
0
14
8
17
0
28
3
0
5
0
16
2
0
1
0
2
7
5
11
3
0
2
11
6
12
7
4
* Each pregnant horn was assumed to contain six native eggs.
t Only trophoblast tissue was seen in three embryos that had invaded into decidualized
stroma in the presence of progesterone and oestradiol.
luminal forms. Provided that these horns were stretched to reduce their
diameters, central necrotic changes were averted.
As native and transferred eggs developed similarly, the results were pooled
for each hormonal treatment given during culture. The proportion of eggs
retained in the progesterone-treated groups was higher than in the nonprogesterone-treated groups (P < 0-05). This difference was due to the low
numbers of eggs remaining in the control uteri, which survived less well than
those treated with hormones. Most of the embryos, however, were alive, and
the proportions of live to dead embryos did not vary between the treatment
groups. Dead embryos were excluded from the following analyses.
FIGURES
1-4
Figs. 1 and 2 are photomicrographs and Figs. 3 and 4 are electron-micrographs of
transferred embryos or parts of embryos, cultured in uteri from ovariectomized mice.
Fig. 1. A blastocyst with enlarged trophoblast cells (T) and endothelium that has
migrated to become distal endoderm (DE). Cultured for 2 days in Trowell's T-8
medium containing 2-5 /tg of progesterone and 0-5 /tg oestradiol per ml.
Fig. 2. A solid embryo with enlarged trophoblast cells (T) that have invaded the
uterine stroma (arrows). The embryonic tissue (EM) is in direct contact with the
trophoblast. Cultured for 3 days in Eagle's MEM containing 2-5 //g of progesterone
per ml.
Blastocyst invasion in mouse uterus in vitro
303
304
P. S. GRANT, I. LJUNGKVIST AND O. NILSSON
Hormone requirements for invasion
Invasion only occurred in the progesterone-treated groups. The first developmental change of native or injected blastocysts was expansion and elongation
of the blastocoele cavity. This was followed by enlargement of the trophoblast
cells (Fig. 1), which occurred in all the media and has already been described
(Grant, 1973 a).
Neither progesterone nor oestradiol (0-05 /ig and 100/tg/ml) had any
discernible effect on the ultrastructure of the epithelial or the stromal cells.
These cells resembled those of the uteri cultured in the control medium.
Uteri cultured in the presence of progesterone, and fixed by immersion
alone, usually had closed lumina. Interdigitation of microvilli from the apposed
luminal cell membranes was, however, sometimes prevented by the presence
of an electron-dense secretion. Conspicuous lumina were present in uteri
cultured in control medium or with the addition of oestradiol alone.
Decidualization and embryogenesis
The stromal tissue did not decidualize in response to invasion or to embryonic
development in the absence of hormonal pre-treatment. Pre-treatment of
ovariectomized, superovulated mice with progesterone and oestrogen allowed
decidualization of the stromal tissue, but only in uteri that already showed
Pontamine blue-positive zones immediately prior to their culture.
In one uterus embryos had invaded a little way into decidual tissue but
decidualization was incomplete, as decidual cells were only present antimesometrially and did not form chambers surrounding these embryos. Three
of five embryos contained only a few trophoblast cells after culture.
Differentiation and growth of the embryonic tissue followed blastocyst
expansion and accompanied trophoblast cell enlargement (Fig. 2). A higher
proportion of eggs had developed beyond the delay stage in the progesteronetreated groups (P < 0-05). Embryonic differentiation often proceeded in an
apparently normal way, until the distal endoderm had extended about half-way
to the abembryonic pole. Thereafter, the yolk cavity collapsed and endodermal
and trophoblast cells intermingled (Fig. 2). This collapse was associated with a
foreshortening of the abembryonic hemispheroidal circumference with bunching
and proliferation of the trophoblast cells. The growth of both the embryonic
and trophoblastic tissues, however, continued and the size of the embryos after
3 days' culture was greater than after 2 days.
The stage of embryonic development was roughly correlated with the degree
of trophoblastic activity. Most eggs that had not developed beyond the proximal
endoderm stage were non-invasive, while embryonic mass development beyond
that of the distal endoderm stage usually accompanied stromal invasion.
Blastocyst invasion in mouse uterus in vitro
305
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Fig. 3. An epithelial cell (E) partially surrounded by invasive trophoblast tissue (T).
Trophoblastic processes have penetrated to loosen this cell from its neighbour,
although both its internal organelles and plasma membrane are undamaged. The
trophoblast has also penetrated between the plasma membranes of the other
epithelial cells (arrows). Culture conditions were as described for Fig. 1.
306
P. S. GRANT, I. L J U N G K V I S T AND O. NILSSON
Fig. 4. The junctional zone between the invasive trophoblast (T) and the stromal
tissue (S). Fibrillar stromal material (F) is present immediately adjacent to the
trophoblast. The embryo was cultured for 3 days in Eagle's MEM containing 2-5 fi%
of progesterone and 005/ig of oestradiol.
Blastocyst invasion in mouse uterus in vitro
307
Table 2. The development of eggs after transfer to hormone-treated ovariectomized
immature mice (numbers of mice and eggs)
Treatment
...
Arachis oil Progesterone
6
Mice
8
70
118
Eggs transferred*
29
31
Eggs found
Trophoblastic activity of the eggs
13
Non-invasive and expanded
6
14
With enlarged cells
14
Invading the stroma
0
It
Embryonic development of the eggs
In delay
22
25
3
With proximal endoderm
7
0
With distal endoderm
1
0
Beyond distal endoderm
0
1
Unknown
1
* 4-13 eggs per horn.
t Invading at the site of injection.
Oestradiol
Progesterone +
oestradiol
10
124
11
11
.153
27
3
6
0
8
24
16
7
1
1
0
2
3
5
1
11
7
The ultrastructure of trophoblast invasion
Three embryos in uteri cultured with progesterone and oestradiol were
studied with the electron microscope. One invading the epithelium had differentiated distal endoderm and two invading non-decidualized stromal tissue had
embryonic development beyond the distal endoderm stage.
The epithelium was invaded by the lateral trophoblast cells on one side of the
embryo. Trophoblastic projections of variable size were positioned between
the epithelial cell membranes (Fig. 3). Partial encirclement of cells by these
projections did not damage the separated cell membranes. Large parts of
epithelial cells containing structurally normal organelles were completely
encircled by the trophoblast in the sectional plane.
The stromal cells in contact with the invasive giant trophoblast cells were
often viable and usually contained cytoplasmic fibrillar material concentrated
immediately adjacent to the trophoblast (Fig. 4). This material often occupied
gaps between the membranes of trophoblastic and stromal cells, especially
around degenerating cells. Only rarely, when this material was absent, were
trophoblastic processes seen between stromal cell membranes.
Lysosome-like bodies and lipid inclusions were more prevalent and larger
in invasive than in non-invasive trophoblastic tissue.
In vivo experiments (Table 2)
The proportions of transferred embryos retained in uteri of the immature
mice were generally low and heterogeneous for the treatment groups (P < 0-001).
308
P. S. GRANT, I. LJUNGKVIST AND O. NILSSON
The hormonal requirements for egg implantation in immature mice
Expanded non-invasive blastocysts with or without enlarged trophoblast
cells were found in all groups but invasive embryos were normally only present
in mice given progesterone and oestradiol. Giant trophoblast cells had invaded
into stromal tissue that had undergone a varied degree of decidualization. The
three largest embryos had small ectoplacental cones. The one embryo that had
invaded in a mouse given progesterone alone did so at the site of blastocyst
transfer. It consisted solely of giant trophoblast cells and had not induced a
decidual reaction in the stroma.
The proportions of non-invasive blastocysts that were expanded and that had
enlarged cells were higher in the progesterone- than in the non-progesteronetreated groups (P < 0-05 in both cases).
Development of the embryonic tissue in immature mice
Proximal endoderm had differentiated in embryos from all groups, but only
in mice given both progesterone and oestradiol did differentiation progress to
the formation of egg-cylinders. These usually had disorganized embryonic
ectoderm with no evidence of proamniotic cavity formation and their shape
and orientation (but not their size) varied according to the degree of accompanying decidualization.
Embryos within large decidual swellings were generally normal in shape,
although a minority were small with disorganized development. Embryos
associated with areas of decidualization that were small relative to their developmental stage were not situated in crypts, but were compressed within
mesometrially displaced lumina and were angular in shape.
DISCUSSION
The hormonal requirements for invasion in vitro
Progesterone was sufficient and necessary for blastocyst invasion in horns
from ovariectomized adult or immature mice cultured in chemically defined
media. Progesterone was important for the continuation of trophoblastic
activity rather than for its initiation, as this occurred in all of the treatment
groups. The only morphological change unique to the progesterone-treated
uteri was luminal closure, which probably aided invasion by providing continued close contact between the trophoblast and the uterine epithelium.
Invasion in vitro probably depended on the inability of progesterone to produce
or maintain a progestational endometrium.
Oestrogen was not required for invasion in vitro, perhaps because there was
no progestational epithelium for it to modify. Nor was the addition of oestrogen
to the culture medium associated with any ultrastructural response, even at
levels that induced changes in cultured calf (0-05/^g/ml; Maurer, Rounds &
Blastocyst
invasion in mouse uterus in vitro
309
Raiborn, 1967) and human endometrium (100 /^g/ml; Gordon, Kohorn, Gore &
Rice, 1973).
Decidualization and embryogenesis in vitro
Trophoblastic invasion and embryonic differentiation were not associated
with decidualization of the stroma in uteri from mature mice. These results
suggest that the failure of invasive embryos to induce decidualization in uteri
from immature mice (Grant, 1973a) was due not to their immaturity but to the
hormonal unresponsiveness of the stromal cells.
The normal pattern of embryogenesis is disturbed during culture: the yolk
cavity collapses, the distal endoderm fails to complete its encirclement within
the trophoblastic shell and Reichert's membrane does not form. The collapse
is probably attributable to several factors. These include the inherent propensity of embryos to collapse in vitro (Gwatkin, 1966; Hsu, 1973; Wilson &
Jenkinson, 1974), the effect of embryonic compression by the uterus and the
failure of formation of an implantation chamber. Decidualization increases
the rigidity of the stroma and results in the formation of a protective chamber
(Snell & Stevens, 1966) that probably expands spontaneously owing to the
degeneration of its innermost layers (Finn & Hinchliffe, 1964). A similar compression was observed in vivo among egg-cylinder-stage embryos in the uteri of
immature mice where normal development of the implantation chamber had
been impeded because of incomplete decidualization. Such embryos were
often orientated along, instead of at right angles to, the direction of the
uterine lumen, suggesting that normal orientation is also dependent on proper
decidualization.
The small decidual swellings that developed in vitro after hormonal pretreatment of the uterine donors were not associated with improved embryogenesis, but with more than usually abnormal development. Decidualization
was confined to one side of the lumen; thus, rather than forming a protective
chamber, it probably aided in the compression of the embryos.
The ultrastructure of invasion in vitro
The epithelium in the present study was usually viable after culture. This
suggests that the trophoblast in vivo could invade viable, hormonally unmodified
epithelium, as well as epithelium degenerating as a consequence of decidualization (Finn & Hinchliffe, 1964).
The morphology of epithelial invasion in vitro appeared similar to that
previously described for invasion in vivo (Finn & Lawn, 1968; Potts, 1968;
Enders & Schlafke, 1969; Tachi, Tachi & Lindner, 1970). The appearance of
structurally normal cells adjacent to the trophoblast in the present work was
also a general feature of invasion in vivo in the six species studied by Enders &
Schlafke (1969).
Maternal collagen-like fibrillar material was observed at the trophoblastic
310
P. S. GRANT, I. LJUNGKVIST AND O. NILSSON
boundary surrounding the two embryos invading the stromal tissue in vitro.
Similar material has been reported in this situation in the rat in vivo (Potts,
1969; Tachi etal. 1970).
We thank Marianne Grubb, Barbro Pettersson and Elisabeth Lindgren for skilful technical
assistance, Athanasius Venardos and Michael Rutberg for the photographs, Brita Sydh and
Ingegerd Mjoback for typing this work. Financial support was provided by the WHO
(contract no. 362-12/70-2000) and The Swedish Medical Research Council.
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ENDERS,
{Received 28 January 1975)