/ . Embryol. exp. Morph. Vol. 25, 2, pp. 165-174, 1971
Printed in Great Britain
165
His to chemical observations on early
implantation in the mouse
By M. S. R. SMITH 1 AND I. B. WILSON 2
From the Department of Zoology, Southampton University,
and Department of Zoology, University College of
North Wales
SUMMARY
Acid phosphatase, polysaccharide complexes and lipids were studied in the uterus and
embryo of the mouse from 90 to 120 hpost coitum (p.c).
The pattern of acid phosphatase-rich vesicles (ERV) in the uterine epithelium and its
manner of degeneration are discussed.
At 90 h p.c. there are large ERV in the inner cell mass indicating degenerating cells;
these are not detectable at 96 hp.c.
A border of lysosomes is prominent in regions of the peripheral trophoblastic cytoplasm
adjoining the epithelium at 96 hp.c. and this may be involved in membrane changes which
facilitate closer contact and interactions between the embryonic and maternal cells.
Discrete aggregates of large ERV are present in the epithelium and trophoblast at sites
scattered around the abembryonic region, from 96 to 104 h p.c.
From 94 to 100 hp.c. macrophage-like cells can be found in the uterine epithelium.
In the later stages of implantation, 110-120 h p.c, the pattern of acid phosphatase-rich
vesicles becomes progressively more complicated with the whole of the epithelium around
the blastocyst gradually becoming degenerate.
INTRODUCTION
In recent papers attention has been drawn to some of the cellular changes
occurring in the blastocyst and immediately surrounding uterine tissues in the
early stages of implantation in the mouse, from about 90 to 120 h post coitum
(p.c.) (Wilson, 1963; Potts & Wilson, 1967; Potts, 1968,1969; Finn & McLaren,
1967; Finn & Lawn, 1968).
Shortly before the loss of the zona pellucida some of the inner cell mass cells
(ICM) die and are apparently phagocytosed by neighbours. The inclusions
so formed are broken down, and so disappear from the ICM, immediately
following zona loss and initiation of implantation (Wilson, 1963; Potts &
Wilson, 1967; Wilson & Smith, 1968, 1970). Simultaneously with the disappearance of inclusions from the ICM, similar, discrete inclusions ('bodies')
1
Author's address: School of Anatomy, The University of New South Wales, P.O. Box 1,
Kensington, N.S.W., Australia, 2033.
2
Author's address: Department of Zoology, University College of North Wales, Bangor,
Caernarvonshire, U.K.
166
M. S. R. SMITH AND I. B. WILSON
appear in the abembryonic trophoblast and the epithelium adjoining it (Wilson,
1963; Finn & McLaren, 1967; Potts, 1968, 1969; Wilson & Smith, 1968, 1970).
It seems likely that most, if not all, of the inclusions seen in the trophoblast/
epithelium region are derived from epithelial cells in various stages of degeneration and phagocytosis by the trophoblast. Trophoblastic giant cells are
not formed for some hours after the first inclusions appear (Wilson, 1963).
Relatively few epithelial cells appear to be involved in this process at this
time (90-100 h p.c), which precedes general epithelial degeneration by 12 h
or more.
We wanted to know more of the factors involved in these particular events
and the present paper is concerned with the distribution of acid phosphatase
in the blastocyst and uterus through the implantation stages. Acid phosphatase
is a convenient 'marker' enzyme which is probably associated with other lytic
enzymes involved in cell death and phagocytosis. Lipids and polysaccharide
complexes were also briefly examined for comparative purposes.
MATERIALS AND METHODS
Females of the CBA strain were mated with males of the A strain to give
A x CBA hybrid embryos. The CBA females were killed at 90-120 hp.c. The
material was fixed in 10% formol calcium (4 °C) for 18 h and then transferred
to sucrose solution at 4 °C. Implantation sites were examined from pregnant
females at the following times, the numbers of females being given in brackets:
90 hp.c. (20), 92-94 hp.c. (40), 94-96 hp.c. (45), 96-100 hp.c. (30), 100-108 h
p.c. (27), 108-115 hp.c. (25), 115-120 hp.c. (18).
The cold-fixed material was sectioned at 7-8 / t o n a microtome cryostat.
Acid phosphatase was localized by the method of Burstone (1958); the optimum
incubation time for the uterine material was 30 min, although incubations of
up to 60 min were used. Control sections were taken at all stages using the
incubation material without the fast garnet azo dye. Lipids were localized by
the sudan black B method of McManus (Pearce, 1960, p. 850) with a slight
modification. Instead of the paraffin-embedded sections, frozen fixed material
was used. Control sections were extracted with acetone before treatment with
sudan black B. The polysaccharide complexes were shown by the periodic
acid-Schiff (PAS) method after McManus (Pearse, 1960, p. 382).
RESULTS
General distribution of acid phosphatase
The period from the loss of the zona to the loss of the uterine epithelium
has been divided, for convenience only, into stages for which approximate
timings are given. For the purpose of this paper the small acid phosphatase-rich
vesicles (< 1 ju) will be called lysosomes and those larger than 1 fi will be
called enzyme-rich vesicles (ERV).
Histochemical studies on implantation
167
(a) 90 h p.c.
At this stage the zona is still present (Fig. 1A). The embryo has numerous
large ERV (> 3 /i) in the ICM which can be distinguished from the smaller
ERV and lysosomes in the cytoplasm of the trophoblast cells. In the uterine
epithelium there are many lysosomes and medium-sized ERV (1-3 /i) in the
supranuclear region, very few in the peri nuclear region and numerous mediumsized ERV in the subnuclear cytoplasm. This epithelial pattern remains 'stable'
until 104 hp.c. The uterine epithelium has many more ERV than the embryo.
Stromal cells show a diffuse pattern of lysosomes, except for the stromal
macrophages and fibroblasts, which have many lysosomes and ERV.
(b) 92-94 h p.c.
The uterine epithelial pattern of lysosomes and ERV is the same as in the
90 h p.c. period but the embryonic pattern starts to change. The zona has
disappeared and the number of large ERV in the ICM begins to fall; the ICM
in Fig. 1C is in the normal mesometrial position whereas in Fig. 1B it is antimesometrial. This is not unexpected at this stage because orientation of the
ICM normally occurs in this period (Kirby, Potts & Wilson, 1967). The first
signs of a clearly defined area of lysosomes concentrated in the trophoblast
cytoplasm applied to the maternal epithelium is observed at this stage (Fig. 1B),
but as yet few trophoblast cells display this activity.
(c) 94-96 h p.c.
The large ERV have mainly disappeared in the embryo and the ICM has
very little acid phosphatase activity. Attachment of trophoblast to maternal
epithelium is marked and the concentration of lysosomes in the trophoblast
at the maternal-embryonic junction is very prominent (Fig. 1D). Some of
the trophoblast cells have large ERV associated with many lysosomes in the
cytoplasm (Fig. 1D).
(d) 96-100 h p.c.
The border of lysosomes in the trophoblast becomes less prominent and
large ERV appear in a number of the cells. An area of abembryonic trophoblast
demonstrates the development of this pattern (Fig. 1E). In one example there
is considerable acid phosphatase activity in the trophoblast and an associated
area of ERV in the uterine epithelium. Other examples of large acid phosphataserich inclusions in the trophoblast appear later in this stage. Fig. I F shows an
accumulation of large ERV. In the stroma, predecidual cells have more lysosomes
than at earlier stages.
(e) 100-108 h p.c.
The embryo has become elongated and the first signs of the distal endoderm,
with some well-defined ERV, are evident. Occasional 'cells' with large ERV
168
M. S. R. SMITH AND I. B. WILSON
are found on the inner aspect of the trophoblast and these may be found in
the period 92-104 h p.c. Large acid phosphatase-rich inclusions are prominent
in the trophoblast. In the uterine epithelium a number of medium and large
ERV appear, particularly in the supranuclear cytoplasm, most noticeably
antimesometrially, and this becomes more pronounced towards 108 h p.c.
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Histochemical studies on implantation
169
(/) 108-115 h p.c.
A major feature of this stage is the degeneration of the uterine epithelium.
The mesometrial uterine epithelium shows a considerable reduction in height
from 12 to 6 ju, on average and large ERV are prominent in the cytoplasm.
Epithelial degeneration is even more pronounced in the antimesometrial region,
with accumulations of large ERV. A major feature of the later period (115 h p.c.)
is the increase in egg cylinder size. Areas of the epithelium have been replaced
by trophoblast, particularly in the antimesometrial region (Fig. 2 A), but the
much-reduced epithelium with large ERV is still prominent mesometrially.
(g) 115-120 h p.c.
The final stage in this study is shown in Fig. 2B, where there is a well-formed
egg cylinder showing a considerable number of lysosom.es in the proximal
endoderm in comparison with the embryonic ectoderm. Most of the uterine
epithelium has disappeared, with only a small degenerate area being found
mesometrially. Adjacent to the trophoblast there are a few discrete areas of
concentrated lysosomes. The decidual tissue forms a prominent mass by this
time.
Specific cells rich in acid phosphatase in the uterine epithelium and stroma
Numerous fibroblasts and macrophages containing ERV and lysosomes are
found in the uterine mucosa prior to decidua formation. During the early
stages of implantation these cells can be found near the basement membrane
of the uterine epithelium (Fig. 1A, D, E). Between 94 and 100 h p.c. a few
examples of macrophage-like cells can be found in the uterine epithelium. The
epithelium looks normal and in the preliminary stages of preparation the
embryo also looked normal but unfortunately it was displaced before final
FIGURE 1
A-F show acid phosphatase activity.
(A) 90 h p.c. Zona pellucida still present. This section shows the typical embryonic
acid-phosphatase pattern and maternal uterine epithelial pattern. 30 min incubation,
x300.
(B) 92-94 hp.c. The zona has disappeared and thefirst.sign of the lysosomal border
is evident in some trophoblast cells. There are large ERV in the antimesometrial
1CM (pointers). 30 min incubation, x 300.
(C) 92-94 hp.c. ICM is in normal mesometrial position and shows the usual pattern
of ERV observed for a few hours around the time of zona loss. 30 min incubation,
x300.
(D) 94-96 h p.c. This shows the prominent border of lysosomes in the trophoblast
near the embryo-maternal junction. There is an obvious reduction in large ERV
in the ICM. 30 min incubation, x 300.
(E) 96-100 h p.c. The lysosome border is less prominent. Build-up of ERV in
some trophoblast cells is obvious (pointers) and in epithelial cells. 30 min incubation,
x300.
(F) 96—100 h p.c. Shows an accumulation of medium-sized ERV in a trophoblast
cell. 30 min incubation, x 300.
170
M. S. R. SMITH AND I. B. WILSON
mounting, so we cannot be certain of its condition (Fig. 2C). There appear
to be three types of cell especially rich in acid phosphatase in, or close to, the
epithelium:
(1) A cell 15 x 5 fi without a prominent nucleus and having many mediumsized ERV or a few large ERV (Fig. 2D).
. v D
Histochemical studies on implantation
171
(2) Round cells with prominent nuclei, superficially resembling large lymphocytes (12 x 10 jit), with few lysosomes (Fig. 2C).
(3) Macrophage-like cells with prominent nuclei and ERV (Fig. 2C).
Periodic acid-Schiff
The PAS technique was used to see whether one could relate the large ERV
and acid phosphatase inclusions to a strongly PAS-positive reaction.
Strongly PAS-positive inclusions are frequently found in the ICM during
the 90-94 hp.c. period (Fig. 2E). These are very similar to the acid phosphataserich inclusions shown in Fig. 1C. The supranuclear cytoplasm of the uterine
epithelium is moderately PAS-positive and in the antimesometrial half there
are, frequently, strongly PAS-positive inclusions (6x4 JLL) inside the basement
membrane. Inclusions superficially similar to the latter appear in the mucosa.
Also there is a moderately PAS-positive area occupying the position of a
uterine epithelial cell shown in Fig. 2E. This is not a frequent occurrence in
the 92-104 h p.c. period. At approx. 104 h p.c. the trophoblast cells have
a number of dark-red, strongly PAS-positive inclusions. These are often
associated with an increase in PAS-positive material in the uterine epithelium.
PAS-positive inclusions are found in most trophoblast cells at about 108 hp.c.
Lipid
At 100-104 h p.c. sudanophilic granules are concentrated mainly in the
supra- and subnuclear cytoplasm of the uterine epithelium. In the embryo the
ICM has little sudanophilic material but the trophoblast cells often have
a number of sudanophilic inclusions in a corresponding position to the PASFlGURE 2
A-D show acid phosphatase activity.
(A) 115 h p.c. Shows an increase in egg-cylinder size and the very degenerate
nature of the mesometrial epithelium. The trophoblast has replaced some of the
epithelium (pointer). 30 min incubation, x 200.
(B) 120h p.c. Shows development of the egg cylinder and prominent proximal
endoderm (arrow). Also prominent are areas of acid phosphatase activity in the
trophoblast and stroma (pointers). 30 min incubation, x 250.
(C) 94-100 h p.c. Shows an example of macrophage-like cells inside the basement
membrane of the uterine epithelium (pointer). There is a large lymphocyte inside the
basement membrane (upper pointer). 30 min incubation, x 350.
(D) 100-104 h p.c. Shows a cell in the epithelium with three large ERV (pointer)
and some in the trophoblast. 30 min incubation, x 150.
(E) 92-94 h p.c. PAS reaction. Shows two PAS-positive inclusions in the ICM.
There are also two strongly PAS-positive inclusions in the epithelium (pointers).
x300.
(F) 96-100 h p.c. Stained for lipid (sudan black B). Shows sudanophilic inclusions
in the trophoblast and the presence of a border of small sudanophilic granules in
a trophoblast cell near the embryo-maternal junction (pointer), x 350.
12
EM B 25
172
M. S. R. SMITH AND I. B. WILSON
positive and acid phosphatase-rich inclusions. A few large trophoblast cells
have a border of sudanophilic granules near the uterine epithelial border
(Fig. 2F).
DISCUSSION
During the period 90-104 h p.c. the epithelium has a similar pattern of acid
phosphatase-rich vesicles to that found at di-oestrous (Smith, 1970). There is
a difference in the lack of large ERV in the supranuclear cytoplasm paralleled
by an increase in lysosomes (small ERV). Between 104 and 110 h p.c. there
is a build-up in the uterine epithelium of large ERV in the supra- and subnuclear
regions and an increase in perinuclear ERV. The process of general epithelial
degeneration appears to commence during this period and continues until
approximately 120 h p.c, with concentration of large ERV and reduction in
epithelial height. In the extreme antimesometrial region there are marked
signs of epithelial degeneration and phagocytosis by the trophoblast from
110 h p.c.
In the blastocyst at 90 hp.c, while the zona pellucida is still intact, numerous
large ERV are found in the ICM. These probably represent the spontaneous
degeneration of embryonic cells, as described by Glucksmann (1951) for
developing systems in general and Potts (1969) and Wilson & Smith (1970)
for mouse blastocysts. The number of ERV per embryo is variable but the
usual situation at the time of zona loss is represented in Fig. 1C. Soon after
zona loss the number of large ERV is considerably reduced and none are
detectable by 96 h p.c. The 'normal' embryonic cell has very few large ERV
and few lysosomes when compared with epithelial cells.
When embryo-maternal contact is well established, at 94 h p.c, in some
trophoblast cells the first signs of lysosomes appear in peripheral cytoplasm
adjoining the epithelium. This 'border' is very prominent at 9 6 h p . c and can
persist until 104h p.c (Fig. ID). During this period the border is often
associated with an increase in acid phosphatase-rich material in the epithelium
either in the form of single large ERV or collections of lysosomes. The next
stage appears to be accumulation of many lysosomes in the trophoblast cytoplasm and this is followed by the appearance of large acid phosphatase-rich
inclusions in the trophoblast which are similar to those present in the epithelium.
Often it is difficult to judge whether an inclusion is in the trophoblast or in
the epithelium, the trophoblast/epithelial interface being difficult to define.
These inclusions appear to be both PAS-positive and sudanophilic, and they
correspond well with electron-dense inclusions seen in electron micrographs
of similar stages (Potts, 1968). The suggestion put forward by Potts (1968),
Finn & Lawn (1968) and ourselves (Wilson & Smith, 1970) that the inclusions
may result from premature death and phagocytosis of discrete uterine epithelial
cells is given further credence by the fact that the trophoblast inclusions are
usually associated with either large ERV or collections of lysosomes in the
Histochemical studies on implantation
173
adjacent uterine epithelium. It is interesting to note the formation of the border
of lysosomes in the trophoblast at the time of trophoblast attachment to the
epithelium. We can only speculate as to its function at this stage but perhaps
it is involved in membrane changes which facilitate closer contact and interactions between the cells. It is possible that this may bring about death of
individual epithelial cells. The final breakdown of individual epithelial cells
in the early stages (96-104 hp.c.) seems to depend upon them being phagocytosed
by the trophoblast; little autolysis seems to occur.
During implantation a number of macrophage-like cells are seen close to
the epithelial basement membrane. From 94 to 98 hp.c. these cells can be found
in the uterine epithelium. Although the epithelium at the implantation site
appears to be normal, the embryo was dislodged during preparation, therefore
speculation about the role of the macrophage-like cells and their effects must
wait until more examples are obtained.
In the later stages of implantation, when general epithelial breakdown sets
in (108-120 h p.c), the pattern of acid phosphatase-rich vesicles becomes
progressively more complicated. Epithelial cells in the extreme antimesometrial
region appear to degenerate earlier than the more mesometrially placed cells
and they are then phagocytosed by the trophoblast. The mesometrial epithelial
cells then diminish in height and accumulate a few large ERV as they degenerate.
Autolysis appears to play a part in epithelial degeneration at this stage, and
the process appears similar to that analysed by Scheib (1964, 1965) in the
regressing Miillerian duct and by Salzgeber & Weber (1966) in regressing
mesonephros of the chicken. When the antimesometrial epithelium has been
replaced by trophoblast at 120 hp.c. discrete areas of lysosomes may correspond
with trophoblast giant cell activity.
'Cells' with large ERV are occasionally seen on the inner aspect of the
trophoblast at any time from 92 to 104 hp.c.; these are probably degenerating
cells, detached from the ICM at the time of loss of the zona pellucida when the
ICM migrates to its mesometrial position (Kirby, Potts & Wilson, 1967), and
in the absence of phagocytic neighbours their final breakdown is delayed. This
type of cell degeneration has been observed in routine histology (I. B. Wilson,
unpublished data).
Finally, the significance of the border of sudanophilic granules in large
trophoblast cells near the uterine epithelium at 100-108 h p.c. has yet to be
ascertained.
RESUME
Observations histochimiques sur les premieres etapes
de Vimplantation chez la souris
La phosphatase acide, des complexes de polysaccharides et les lipides ont ete suivis dans
Puterus et l'embryon de souris entre 90 et 120 h post-coitum (p.c).
Nous avons discute la distribution des vesicules riches en phosphatase acide (ERV) dans
l'epithelium uterin et la maniere dont elles degenerent.
174
M. S. R. SMITH AND I. B. WILSON
90 h apres l'accouplement, il y a de grandes ERV dans la masse des cellules internes
indiquant l'existence des cellules en degenerescence; celles-ci ne sont pas decelees 96 hp.c.
Dans le trophoblaste peripherique, une bordure de lysosomes est tres apparente dans les
regions cytoplasmiques avoisinant l'epithelium 96 h p.c.; ceux-ci peuvent etre impliques
dans les changements membranaires qui permettent des contacts plus etroits et des interactions
entre les cellules embryonnaires et maternelles.
Des agregats discrets de grandes ERV sont presents dans l'epithelium et le trophoblaste
et dissemines autour de la region embryonnaire entre 96 et 104 h apres l'accouplement.
Entre 94 et 100hp.c. des cellules ressemblant a des macrophages peuvent etre observees
dans l'epithelium uterin.
Dans les stades plus tardifs de l'implantation, 110-120 hp.c, la distribution des vesicules
riches en phosphatase acide devient progressivement plus compliquee tandis que tout
l'epithelium autour du blastocyste degenere.
We are grateful to Dr Doreen Pugh of Southampton University for her advice and criticism
of this work. One of us (M.S.R. S.) is particularly indebted to the Wellcome Trust for the
award of a Research Fellowship during tenure of which this work was undertaken.
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{Manuscript received 30 April 1970, revised 14 August 1970)