/. Embryol. exp. Morph. Vol. 28, 2, pp. 279-312, 1972
279
Printed in Great Britain
An investigation of inner cell mass
and trophoblast tissues following their isolation
from the mouse blastocyst
By R. L. GARDNER 1
With an Appendix by M. H. JOHNSON 1
From the The Physiological Laboratory, Cambridge
SUMMARY
1. Inner cell mass (ICM) and trophoblast were isolated from 3^-day post-coitum mouse
blastocysts by microsurgery.
2. Trophoblastic fragments formed vesicles in culture but did not aggregate with other such
fragments. They proved as effective as intact blastocysts in inducing decidua in recipient uteri,
but thereafter failed to proliferate.
3. Isolated ICMs remained as solid balls of cells that readily aggregated in pairs or groups
in culture but failed to induce implantation changes in receptive uteri.
4. Possible explanations for the failure of isolated trophoblast to proliferate after implantation are discussed. It is argued that presence of ICM tissue is necessary for trophoblast
proliferation, and suggested that the ICM exerts its effect by controlling development of the
ectoplacental cone.
INTRODUCTION
One of the earliest differences to arise between cells of the mammalian embryo
is evident at the blastocyst stage with formation of the trophectoderm and inner
cell mass (ICM). Attention has been devoted to establishing whether this
differentiation is determined in the egg prior to division (Dalcq, 1957; Seidel,
1960; Mulnard, 1965) or by the relations between naive blastomeres during
cleavage (Mintz, 1965; Tarkowski & Wroblewska, 1967; Graham, 1971). The
question of the particular properties that serve to distinguish the two tissues in
the early blastocyst has so far been largely neglected.
Earlier studies involved disaggregation of morulae and blastocysts of both the
rabbit and mouse, and culture of the cells isolated from them (Cole & Paul, 1965;
Cole, Edwards & Paul, 1965). Since no attempt was made to separate ICM and
trophoblast tissue prior to disaggregation, the cells could not be assigned unequivocally to one or other type.
The present study was thus aimed at separating the blastocyst into its constituent tissues, and comparing their properties both in culture and after transfer
to the uterus. This project promised to provide criteria for distinguishing these
1
Authors' address: Physiological Laboratory, Cambridge, CB2 3EG, U.K.
280
R. L. GARDNER AND M. H. JOHNSON
Table 1. Viability of blastocysts transferred after 8^—11 h at room temperature in
phosphate-buffered M199 + 10% serum medium
(Recipients allowed to deliver young.)
Blastocysts
Recipient code
R4 (24. 10. 69)
R3 (6. 11.69)
R 3 (16. 10. 69)
R 4 (16. 10. 69)
Hours in vitro
No.
at room
transferred
temp.
bilaterally
8|
9
11
11
No. of
young born
8
10
6
7
10
12
5
8
Comments
—
1 dead,
uncertain
if still-born
—
—
A further three recipients did not become pregnant.
tissues in investigations into the mechanism of their differentiation, and to
clarify their roles in implantation and interrelations in later development. The
mouse was chosen because it is the species used in most studies of early mammalian development and because its blastocyst is very resilient to microsurgery
(Gardner, 1968, 1971, 1972; Lin, 1969). A preliminary report of part of this
work has been published elsewhere (Gardner, 1971).
Previous accounts of the handling and transfer of so-called 'pure' trophoblast
of murine origin have dealt exclusively with the ectoplacental cone of the implanted egg-cylinder (Grobstein, 1950; Billington, 1965; Simmons & Russell,
1966; Clarke, 1969). Despite the enveloping cell layer of the unimplanted blastocyst being called trophoblast its relationship with the ectoplacental cone is
uncertain. This note of caution is justified by the following results.
MATERIALS AND METHODS
Preparation of donor females and recovery of blastocysts
Adult or 3- to 4-week-old PDE random-bred or Er inbred albino mice were
superovulated by intraperitoneal injection of pregnant mares' serum gonadotrophin (PMSG - Gestyl, Organon) followed 48 h later by human chorionic
gonadotrophin (HCG-Pregnyl, Organon). Five i.u. of each hormone were given
to adults and 10 i.u. to the immature female mice. PDE females were paired
with males of the same or CBA/H-T6 inbred strain, and Er females exclusively
with CBA/H-715 males after administration of HCG. Females with plugs were
killed and their excised uterine horns flushed for blastocysts either between
96-102 or 117-122 h after HCG. Since no consistent differences were noted
between material obtained at opposite extremes of each time range, blastocysts
Inner cell mass and trophoblast tissues
281
Fig. 1. Schematic side view of hanging drop in manipulation chamber to show
arrangement of blastocyst and fragment of razor blade for sectioning blastocyst to
obtain pure trophoblast. c.s. = coverslip, i.c.m. - inner cell mass, r.b. = fragment
of razor blade, s.p. = suction pipette.
recovered during the former interval (and tissue derived from them) will be
called 3^-day and those recovered during the latter interval will be called 4^-day
blastocysts or fragments.
Conditions for storage, manipulation and culture of blastocysts and fragments
The special conditions imposed by microsurgery required modified methods
for keeping blastocysts in vitro. The medium could not be maintained in equilibrium with an atmosphere of 5 % CO 2 in air. Also, attempts to perform manipulations at 37 °C were unsuccessful because of the great increase in adherence of
cells and debris to the instruments at higher temperature. Manipulations were
thus carried out at room temperature in medium M 199 plus 10 % inactivated
foetal calf serum (Microbiological Associates Inc., or Flow Laboratories,
Scotland), which contained 100 i.u./ml of sodium benzyl penicillin and was
buffered at pH 7-0 with 5-8 % (v/v) Sorensen's phosphate buffer (M/15). TO
avoid unnecessary osmotic shocks or changes in pH this medium was used
throughout. Under these conditions intact blastocysts remained viable for up
to 11 h at room temperature (Table 1).
Microsurgery
A Leitz micromanipulator was used. Blastocysts were placed in a drop of
medium hanging from the coverslip of a chamber (Puliv, Leitz) filled with heavy
liquid paraffin. Microsurgery was carried out at x 125, x 300 or x 500 using
bright-field or Heine phase-contrast observation in a Leitz Laborlux microscope.
Trophoblast tissue was obtained free from ICM as follows. Expanded 3^-day
blastocysts surrounded by intact zonae were orientated and held by suction
pipette against the underside of the coverslip of the hanging drop (Fig. 1). A piece
of the cutting edge of a safety razor-blade attached to a mounted needle with
282
R. L. GARDNER AND M. H. JOHNSON
Residual trophoblast
discarded
Kquau
lYasiment
Maximal trophoblast
fiat: me nt
4- - A
Hquatorial inner cell
mass fragment
Minimal inner eel
mass fragment
Dissected inner
cell mass
Fig. 2. Scheme of manipulations carried out on the mouse blastocyst to obtain
trophoblast and ICM tissue. D, Trophoblast tissue; • , inner cell mass tissue.
(From Gardner, 1971, with permission of F. Vieweg & Sohn.)
Araldite (CIBA) was arranged vertically on one manipulator unit so that its
cutting surface was parallel with the coverslip of the chamber (Fig. 1). By
raising the blade slowly the living blastocyst could be severed, parallel to the
surface of the ICM, either equatorially (to yield equatorial trophoblastic
fragments, Fig. 2 A) or close to the embryonic pole (to yield maximal trophoblastic fragments, Fig. 2B). Sectioning blastocysts through the equator presented
no difficulty. The embryo tended to rotate away from the advancing blade when
it was cut towards one pole. This could be prevented by increasing suction or by
using a needle to help immobilize the embryo. Provided the fragment of razorblade had been cleaned, siliconed and correctly aligned, blastocysts could be
severed with only occasional recourse to the needle to clear one or other fragments from the blade. The choice of well-expanded blastocysts enabled trophoblast to be obtained with little risk of inclusion of ICM cells.
Preliminary attempts to isolate ICM tissue from blastocysts by various
chemical and enzymatic treatments were unsuccessful. ICMs were thus obtained
by penetrating immobilized expanded blastocysts from opposite sides with a pair
of fine, siliconed glass needles, tearing the trophoblast open and pinning it out
as a sheet against the coverslip. The exposed ICM was then gently scraped from
the trophoblast, but was only used if the sheet of overlying trophoblast left
behind was intact. It often proved possible to isolate ICMs relatively free
from cells of the enveloping trophoblast. However, the procedure extensively
damaged the corresponding trophoblast, so comparison between trophoblast
and ICM tissues from the same blastocyst was not possible.
Inner cell mass and trophoblast tissues
283
Culture of trophoblastic fragments, isolated 1CMs and intact blastocysts
Trophoblastic fragments and intact blastocysts were cultured either in hanging
drops in the oil chambers used for microsurgery, or in microdrops on siliconed
coverslips placed in culture dishes (Falcon Plastic, 60 x 15 mm) filled with heavy
liquid paraffin. Isolated ICMs were generally very small and delicate and were
therefore left in the manipulation chambers for culture. Fusion was encouraged
by pairing fragments with a blunt glass needle, and with the warm stage of
the microscope set at 37 °C. This allowed firm contact to be made between
fragments before they were moved to the incubator.
Cell counts on blastocysts and isolated ICMs
The material was prepared according to the air-drying method of Tarkowski
(1966).
Transfer of tissue and blastocysts to the uteri of recipient mice
The 3|-day blastocysts, trophoblastic fragments or isolated ICMs were
transferred to the uteri of mice on the third day of pseudopregnancy, except in
one case where a group of trophoblastic fragments were transferred to a recipient
on the fourth day. The recipients were albino PDE or Er mice mated with
vasectomized males. All ICMs and some trophoblastic fragments were transferred unilaterally, and intact blastocysts introduced into the contralateral horns
served as a control for uterine reactivity. Uterine transmigration of embryos is
a possible complication in such experiments (Runner, 1951; McLaren &
Michie, 1954). However, it was not found among 36 foetuses examined following
transfer of blastocysts of pigmented and albino genotypes to opposite horns
of 6 recipients, nor among 63 implants developed from blastocysts transferred
unilaterally to a further 18 recipients (unpublished observations).
Induction of deciduomata
Intraluminal injection of arachis oil between 15.00 and 16.00 h on the fourth
day of pseudopregnancy was employed for this purpose (Finn, 1965).
Examination of the transferred tissue
Recipient females that received trophoblast tissue were examined between
3 and 8 days after transfer. Horns carrying decidual swellings were fixed in
Sousa or Bouin's fixative. The dehydrated tissue was cleared in supercedrol
(G. T. Gurr), embedded in paraffin wax and serially sectioned at 6-10 jam. The
sections were stained with haemalum and eosin and mounted in DPX (G. T.
Gurr).
Females receiving ICM tissue were given i.v. pontamine sky blue 6 BX (G. T.
19
EMB 28
284
R. L. GARDNER AND M. H. JOHNSON
Gurr) approximately 48 h after transfer of the tissue to determine whether implantation changes had been initiated (Psychoyos, 1961; Finn & McLaren, 1967).
Deciduomata and implants derived from intact blastocysts were processed
histologically as above.
Assessment of implantation rates by counting decidual swellings-complicating
factors
More than one implant may occur within a single decidual swelling (author's
unpublished observations). This phenomenon would not be detected in cases
where derivatives of the ICM are poorly represented or totally lacking (as in
nearly all implants obtained following transfer of trophoblast). Counts of
decidual swellings would thus tend to underestimate the rate of implantation.
Decidualization can also occur from the inevitable trauma accompanying the
transfer operation, and inclusion of these swellings would lead to an overestimate
of the implantation rate. However, in two cases where such deciduomata were
identified with certainty, i.e. in horns where all transferred blastocysts had given
rise to decidual swellings containing embryos or embryonic tissues, there was no
question of their being confused with normal decidua. They were found at the
oviducal end of their respective horns (i.e. at a point corresponding to the
transfer site) and were less than half the normal diameter. One was sectioned, and
was not only completely without embryonic tissues but very poorly orientated
in relation to the mesometrial-antimesometrial axis of the uterus. During this
and a succeeding study (in preparation), further examples of probable traumatic
deciduomata were encountered that showed all the above features, particularly
the small size and poor orientation. Only once did a probable deciduoma
approach the size of an adjacent decidual swelling. Yet even here the orientation
was atypical, being exactly perpendicular to the mesometrial-antimesometrial
axis. So, although these possible complications must be borne in mind their
effect on the results will be slight.
RESULTS
1. Observations on trophoblastic fragments in culture
The following remarks apply to both maximal and equatorial fragments
(Fig. 2 A, B) unless qualified, and are based on study of 139 maximal and 34
equatorial fragments.
Initially, the fragments were solid balls of cells with no visible cavity (Fig. 3 A).
When placed in culture the majority sealed and began to cavitate within 30 min,
becoming obviously vesicular within l ^ h (Fig. 3B). In a typical series 85%
(56/66) cavitated. The remaining 10 may have been damaged during cutting or
handling. Although the vesiculated fragments could cavitate a second time if
torn open, they did not swell to the same size as intact blastocysts.
Twenty-four maximal fragments were placed in contact in 12 pairs and the
285
Inner cell mass and trophoblast tissues
Fig. 3. A pair of maximal trophoblastic fragments (A) immediately after being placed
in contact at 37 °C and (B) 4 h later. The area of contact between the two fragments
is limited, and there is no tendency for them to fuse together.
19-2
VII
VI
V
IV
III
II
Experimental
series
3^-day ICMs of different
genotype - groups
4^-day ICMs from T.T.
blastocysts of same
genotype - pairs
4^-day ICMs from nonT.T. blastocysts of same
genotype - pairs
4^-day ICMs from nonT.T. blastocysts of same
genotype - groups
1 PDE + 1 CBAHT 6 x PDE
3i-day ICMs of different
genotype - pairs
3^-day ICMs of same
genotype - groups
4 PDE
3Er
2 CBAHT6xEr
2CBAHT6xPDE
2 PDE
2Er
2PDE+2CBAHT
3 PDE
2 fused PDE + 2 fused PDE*
3 CBAHT6xPDE
3 fused CBAHT6 x PDEf
(above) + 2 fused
CBAHT6x PDE
2 PDE
2CBAHT6xPDE
No. and genotype
3^-day ICMs of same
genotype - pairs
Category and/?.c. age
Blastocyst fragments placed in contact
Under 7
6
1
1
1
1
Under 8
4
Under 6
—
Under 10i
2 +
4
1
1
7
1
15
14
5
1
1
7
17
18
Post-cultural
morphology
No. of
Approx. time
pairs or
for successful
groups
No.
No.
in series fused not fused fusion (h)
1 failure = ?
2/3 ICMs = T.C.
1 failure = T.C.
2 failures = T.C.
1 failure = T.C.
3 failures = ?
Comments
Table 2. Summary of results ofculturing various categories of fragments of mouse blastocysts in contact at 37 °C
O
o
a
S
d
o
o
ON
oo
i
4-Hay and 3i-day ICMs
of different
genotypes - pairs
Category and p.c. age
A
+ 1 , 3^-Er
l,4iPDE(T.T.) + 1 ,
3iCBAHT6xPDE
2 CBA x PDE
2 PDE
1, 4£CBAHT6 x PDE (T.T.)
No. and genotype
Blastocyst fragments placed in contact
A
r
a
8
1
4
1
4
—
4 or under
4i
3 failures = T.C.
1 failure = ?
Comments
IX
?
—
13
2
3-^-day minimal ICM
11
—
—
4
4
fragments of same
genotype - pairs
—
—
—
12
12
2CBAHT6xPDE
3^-day maximal
X
trophoblastic fragments
of same
genotype - pairs
Abbreviations: T.T. = blastocysts in which abembryonic polar trophoblast had undergone giant cell transformation (Dickson,
1963). T.C. = contaminated with trophoblast cells. This was inferred from the fact that 1 or more peripheral cells had vesiculated,
or because a small eccentric cavity had developed.
* Two already fused pairs of ICMs derived from Series 1.
f A group of three and an already fused pair placed in contact.
series
VIII
lllCIlldl
Experi-
Post-cultural
No. of
morphology
pairs or (
Approx. time
f7"\t" cnr'( *^ ccfi I 1
"No
T\Tn
.LNU.
in series fused not fused fusion (h)
Table 2. (cont.)
oo
<~»
a
a
SO
fv
1
^ ^
* W
for equivalent time
Total
CBAHT6xPDE
PDE
A. Maximal trophoblastic fragments
Total
B. Equatorial
trophoblastic
fragments
Total
C. Fused pairs
of ICMs
Total
D. Intact blastocysts, in vitro
PDE
CBAHT6xPDE
PDE
CBAHT6xPDE
PDE
PDE
CBAHT6xPDE
Genotype
A
1SSue
Category
T")nr
52
14
26
40
12
5
13
30
9
8
17
32
20
No.
transferred
PDE
PDE
PDE
PDE
Er
PDE
Er
PDE
PDE
Genotype
No of
10
2
3
5
1
1
1
3
2
2
4
6
4
11
3
5
8
2
1
2
5
2
2
4
7
4
21
21
21
21
21
31
21
21
21
42
12
14
26
5
1
2
8
0
0
0
25
17
No. of recipient Days p.c. decidual
females
horns at transfer swellings
"Mr» r»f
1NU. \JL
Uterine foster-mothers
(Only recipients with one or more implants included.)
Table 3. Implantation of trophoblast and ICM tissue of 3\-day mouse blastocysts in the uteri of pseudopregnant mice
o
00
o
O
>—i
>
iTl
d
oo
OO
Inner cell mass and trophoblast tissues
289
members of each pair held together at 37 °C until they adhered. These pairs of
fragments remained discrete despite incubation for up to 14 h, though usually
cavitating and showing a limited area of mutual adhesion (Fig. 3 A, B). In no case
was fusion or aggregation observed, and members of a pair could easily be
separated with a glass needle (Table 2, row X).
2. Fate oftrophoblastic fragments transferred to the uteri of pesudopregnant mice
Sixty-five maximal fragments which had been cultured for 2-5£ h were transferred into seven recipients. Thirty-three equatorial fragments, of which only
five had been cultured for 2 h, were transferred into a further four recipients.
Implantation was assessed by looking for discrete decidual swellings, and only
females with one or more such swellings have been included in Table 3. A proportion of both the maximal and equatorial trophoblastic fragments had
implanted.
All but one of the 34 implants examined 4-7 days after transfer of trophoblast
appeared similar (Table 4A, B). Embryo, amnion, allantois, yolk sac, Reichert's
membrane and ectoplacental tissue were absent. A chamber, corresponding
roughly to the position the embryo would occupy in a normal implant, had
almost invariably formed, apparently by pycnosis of the central decidual cells.
Occasionally it had a somewhat spongy or reticular appearance due to the
persistence of a network of elongated cells (Fig. 4). In other decidua the chamber was virtually acellular, and filled with an homogeneous ground substance
broken up by a complex fibrin-like network (Fig. 5), or even with maternal blood
cells. A second consistent feature, particularly of the older implants, was a compact mass of eosinophilic material of unknown origin and composition which
lay between the mesometrial border of the chamber and the zone rich in maternal
sinusoids (Figs. 4, 5). This material sometimes attained large dimensions and
small dispersed foci could occasionally be seen in younger implants. Polymo rphonuclear leucocytes were present in and around it.
The third distinctive feature was the presence in three-quarters of the implants
of cells indistinguishable from the trophoblastic giant cells that surround
normal conceptuses. Their presence constituted the only difference in cellular
composition between decidua induced by trophoblast and deciduomata evoked
by oil. The number of giant cells was similar between 4 and 7 days after transfer
and was trivial compared with the number found in normal implants of equivalent ages (Table 4A, B; Noyes, 1959; Snell & Stevens, 1966). Some of them
were pycnotic, though viable ones persisted in some of the older implants
(Fig. 6).
The single exceptional implant consisted of a small yolk-sac-like structure
surrounded by a thickened Reichert's membrane, and was framed by a network
of healthy giant cells (Fig. 7). This implant was presumably derived from a fragment contaminated with ICM tissue.
The transferred trophoblast thus only gave rise to a very few giant cells which
6
7
6
7
8
8
9i
10i
Hi
Hi
4
5
9i
10i
7i
8i
3||
6
6
12
23
3
1
7
3
6
6
10
—
6
(small
chambers)
23
3
2
8
—
3
1
6
1
—
1
6
6
11
20
2
—
—
5
5
3
3
2
15
1
—
1-6
—
—
1
—
2
—
1
—
7-12
—
—
—
—
—
—
If
13-18
—
—
2§
—
1$
—
Abundant
No. of implants according to no.
of trophoblastic giant cells/implant
* This excludes obvious deciduomata.
t Thirteen out of 14 giant cells degenerating.
X Derivatives of the ICM present (Fig. 7).
§ One was morphologically normal foetus (Fig. 8 A, B).
il These three implants found in recipient R2 (14. 5. 70) in which intact blastocysts had failed to implant (see Table 5).
B. Maximal fragments
only transferred
to recipients
C. Maximal trophoblastic
fragments to one horn
of each of a series of
recipients receiving
intact blastocysts in
other horn
D. As C but where all
intact blastocysts had
failed to implant
A. Equatorial fragments
only transferred to
recipients
Experimental group
Age of
implants
in days
p.c.
Period
in days
between
No. with
transfer Total no. No. with eosinoand
philic
of
central
autopsy decidua* chamber
mass
Table 4. Summary of main features of uterine implants developed from trophoblastic fragments
O
o
d
tn
o
O
>
r
Inner cell mass and trophoblast tissues
291
Fig. 4. Centre of a decidual swelling 6 days after the transfer of maximal trophoblastic fragments. A large mass of eosinophilic material occupies the top centre of the
field. A single pycnotic trophoblast giant cell can be seen in the middle towards the
antimesometrial end of the implantation chamber.
292
R. L. GARDNER AND M. H. JOHNSON
Fig. 5. Another decidual swelling from the same recipient that carried the decidual
site shown in Fig. 4. A large eosinophilic mass lies mesometrially as before. However,
the chamber is larger than the previous one and is virtually free of cells.
Inner cell mass and trophoblast tissues
Fig. 6. Centre of a decidual swelling 7 days after transfer of maximal trophoblastic
fragments. A rather loosely arranged eosinophilic mass occupies most of the centre.
The arrow indicates the position of a viable trophoblastic giant cell.
293
294
R. L. GARDNER AND M. H. JOHNSON
Fig. 7. Exceptional trophoblastic implant 7 days after transfer of maximal trophoblastic fragments. Note the abundant giant cells, absence of ectoplacental cone, and
the central vesicle consisting of a thick Reichert's membrane lined presumably with
cells of the distal endoderm. (Mesometrium towards the top of the figure.)
Inner cell mass and trophoblast tissues
295
were never observed in division. One explanation for the failure of the trophoblast to proliferate might be that the endocrine status of the recipients differs in
these experiments from that of normal pregnancy. Hence the following experiments were undertaken to determine whether trophoblastic fragments developed
similarly in recipients made pregnant by simultaneously transferring intact
blastocysts.
3. Transfer of trophoblastic fragments and intact blastocysts to opposite horns of
pseudopregnant mice
Thirty-six maximal trophoblastic fragments were transferred unilaterally to
six females, and 5-6 blastocysts of the same age and genotype were injected into
the contralateral horns. Five recipients had implants at autopsy. It was found
that trophoblastic fragments devoid of ICM tissue are as effective decidual
stimuli as whole blastocysts (Table 5 A).
All implants developed from intact blastocysts contained foetuses. With two
exceptions, the form of the trophoblastic implants was similar to those described
previously as regards central chamber, eosinophilic mass and scarcity of
trophoblastic giant cells (Table 4C). The absence of giant cells from most of the
oldest implants in this series suggests that death or dispersal of these cells may
occur 7-8 days after transfer.
The two implants which were not typical of transferred trophoblastic fragments were adjacent in one horn of a recipient killed 8 days after transfer. One
contained a well-developed foetus and the other a mass of giant cells (Fig. 8 A, B).
The former might have been due to regulation by a fragment from which all
ICM cells had not been excluded or to accidental transfer of a blastocyst. It was
probably responsible for the occurrence of the second exception. Thus the giant
cells of the first implant were not enclosed by viable decidual tissue on the side
bordering the second implant, since a wide track of entirely pycnotic decidual
tissue extended to the centre of the latter (Fig. 8B). Though no giant cells were
noted actually in the boundary zone they were close enough on both sides to
suggest very strongly that they had invaded the second implant rather than having
originated there.
One recipient had implants in the experimental horn only (Table 5) and these
conformed to the typical pattern (Table 4D).
In a further experiment a single recipient (Rx (29. 5. 70), Table 5B) was killed
only 3 days after transfer of blastocysts to one horn and trophoblastic fragments
to the other. Two implants found in the experimental horn already appeared
abnormal (Fig. 9A-C). Each consisted of a small localized net or cord of
trophoblast cells near the antimesometrial end of a uterine crypt. The uterine
epithelium was missing immediately to either side of them. Neither possessed
a cavity which, since all the control implants did and the entire tract was
fixed as one, is unlikely to have been an artifact of fixation. One implant
contained 25 cells and the second roughly 36 (one or two sections being folded
PDE
PDE
PDE
PDE
Er
R2 (14. 5. 70)
R3 (14. 5. 70)
Rx (18. 5. 70)
R2 (18. 5. 70)
Rx (29. 5. 70)
Right
Left
Right
Left
Right
Left
Right
Left
Right
Left
Right
Left
Uterine horn
MTF
B'cyst
MTF
B'cyst
B'cyst
MTF
MTF
B'cyst
B'cyst
MTF
MTF
B'cyst
Type
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
CBAHT6xPDE
PDE
PDE
Genotype
5
6
6
6
6
7
6
6
n
H
H
H
1*
H
H
ferred
No.
period (h)
H
H
* d = traumatic deciduoma. MTF = maximal trophoblastic fragment. B'cyst = blastocyst.
PDE
Rx (14. 5. 70)
A
B
Genotype
Code
Recipient
Exp.
Group
Transferred tissue
(Only recipients with one or more implants have been included in this table.)
6
6
"No
5
5
3
0
6
7
6+ld*
6
6+ld*
6
2
6
decidual
sites at
autopsy
Table 5. Implantation of maximal trophoblastic fragments in the uteri of pseudopregnant mice that received
intact blastocysts in the contralateral horn
ON ON
o
GO
o
o
vo
Inner cell mass and trophoblast tissues
297
Fig. 8. Two atypical implants obtained following transfer of maximal trophoblastic
fragments to a recipient carrying intact blastocysts in the contralateral horn. (A) Low
magnification to show relationship between them. (B) Detail of region of contact.
298
R. L. GARDNER AND M. H. JOHNSON
Fig. 9. Transverse sections of decidual swellings 3 days after transfer of maximal
trophoblastic fragments and intact blastocysts to opposite horns of the same
recipient. (A) Implanted maximal trophoblastic fragment consisting of only 25 cells,
of which four can be identified in the figure. (B) The second implanted maximal
trophoblastic fragment consisting of approximately 36 cells of which 4-5 may be seen
in the figure. (C) Implanted intact blastocyst in the contralateral horn.
Inner cell mass and trophoblast tissues
299
Table 6. Number of cells in intact 3\-day bias tocysts
and dissected ICMs of PDE mice
Type of embryonic
material
Number scored
Blastocysts
Dissected ICMs
18
10
Number of cells
(mean ± S.E.)
57-3 ±3-7
14-7 + 0-7
100pml
Fig. 10. A single isolated ICM from a 3^-day blastocyst approximately 2\ h after the
beginning of culture. It shows neither cavitation nor intracellular accumulation of
fluid (cf. Fig. 3B). The smaller fragment is residual trophoblastic debris left behind
following dissection of the ICM.
in the latter case). The nuclear diameter of these cells was much less than that of
fully differentiated giant cells. Their cytoplasmic detail was poor and the viability
of some was questionable. No dividing cells were present. Indeed, the number
of cells did not exceed that expected in maximal fragments at the time of transfer
(Table 6).
EM
n 28
300
R. L. GARDNER AND M. H. JOHNSON
Fig. 11. Stages in the fusion of ICMs in vitro. Two pairs of 3^-day dissected ICMs
that had already fused were placed in contact at time zero. (A) +\ h, (B) + \\ h,
(C) + 2 h, (D) + 4 | h after contact (darkground illumination). (From Gardner, 1971,
with permission of F. Vieweg & Sohn.)
4. Observations on ICMs isolated from S\- and 4\-day blastocysts in culture
ICMs were very variable in shape after they had been scraped from the sheet
of overlying trophoblast cells. They rapidly became spherical when cultured, but
remained typically as solid balls of cells without developing a central cavity
(Fig. 10). When placed in contact in pairs or small groups they almost invariably
aggregated to form unitary structures of spherical or near spherical appearance
(Fig. 11). Such fusion consistently took place between pairs of 3^-day ICMs of
Inner cell mass and trophoblast tissues
301
100/mi
Fig. 12. Pair of 3^-day dissected ICMs which failed to fuse after 5 h culture. Intracellular accumulation of fluid, suggesting contamination by trophoblast cells, can be
seen in both fragments.
the same or different genotype (Table 2, rows I and II). Fusion was also observed
between pairs of 4^-day ICMs despite the endoderm having delaminated in
some of these older embryos as discerned by phase-contrast microscopy (Table 2,
rows V, VI and VII). In several instances pairs of ICMs differing by nearly 24 h
in age behaved similarly (Table 2, row VIII).
Where fusion failed the fragments remained discrete but connected by a narrow
waist. Failure was often accompanied by the development of a small eccentric
cavity or by accumulation of fluids within peripheral cells (Fig. 12). Both these
phenomena suggest that the ICM tissue was contaminated with trophoblast
cells. This interpretation is in accordance with the finding that minimal ICM
fragments which retain the cells overlying the ICM (see Fig. 2 B) did not aggregate
under similar conditions (Table 2, row IX). However, minimal ICM fragments
contain more cells than dissected ICMs and hence fusion might simply depend
on cell number rather than specifically on cell type. Cell number is probably not
the limiting factor because pairs of fused ICMs can fuse with other ICMs,
302
R. L. GARDNER AND M. H. JOHNSON
thereby yielding structures containing as many cells as entire blastocysts (Table 2,
row III; Table 6; Fig. 11).
5. Transfer offused pairs of 3\-day isolated ICMs to the uteri of recipient mice
A series of fused pairs of ICMs were transferred unilaterally, intact blastocysts
being placed in the opposite horn of each recipient. The results show that of
17 pairs transferred to females with pontamine blue sites in their control horns
at autopsy, none induced decidual changes (Table 3C). Also, in no case could
the transferred tissue be identified in uterine flushings.
DISCUSSION
The trophoblast and ICM of the mouse blastocyst are very different. The
ability to accumulate or pump fluid (Gamow & Daniel, 1970) and to induce the
decidual changes characteristic of implantation are properties peculiar to the
trophoblast. Though the trophoblastic fragments can form vesicles, as do
cultured fragments of rabbit blastocysts (Daniel, 1961,1963; Klinger, Kosseff&
Plotnick, 1971), they do not aggregate with similar fragments or vesicles. Likewise, mouse blastocysts cannot be induced to fuse together under conditions that
favour fusion of cleaving eggs or morulae in mice (Mintz, 1965; Mulnard, 1971).
This may be related to the presence of specialised junctions between trophoblast
cells (Enders & Schlafke, 1965).
Isolated ICMs resemble cleaving eggs in the ease with which they fuse together,
as also in their inability to accumulate fluid and evoke decidual reactions (Kirby,
1970). The ICMs were fused in pairs before transfer to the uterus to ensure they
contained a similar number of cells to trophoblastic fragments. Hence their
failure to induce decidual changes could have been due to reduction in viability
during the culture period necessary for fusion. Cole & Paul (1965) have argued
that presumptive ICM cells show poor survival in culture. However, this explanation is inappropriate here because cultured ICMs transferred into the
cavity of blastocysts or trophoblastic vesicles can contribute extensively to the
resulting (chimaeric) offspring or give rise to morphologically normal foetuses
respectively (Gardner, 1971).
The cells overlying the ICM cannot be studied directly with present techniques
because they are either removed together with the ICM (in minimal ICM fragments - see Fig. 2B) or destroyed when the ICM is scraped from the blastocyst
(Fig. 2C). Nevertheless, failure of minimal ICM fragments to fuse together
while isolated ICMs do, indicates that these cells differ from those of the ICM.
Whether they are identical to those of the rest of the trophectoderm with which
they are continuous is uncertain at present.
The absence of embryo, amnion, allantois, yolk sac and Reichert's membrane
from implants developed from pure trophoblast accords with the conclusions of
histological studies which attribute an ICM origin to these structures (Snell &
Inner cell mass and trophoblast tissues
303
Stevens, 1966). The similarity of all but 3 of the 61 trophoblastic implants
examined 4-8 days after transfer attests to the homogeneity of the tissue transferred. The very few giant cells evident in the majority of implants are almost
certainly of embryonic rather than maternal origin (Fawcett, Wislocki & Waldo,
1947; Snell & Stevens, 1966; Gwatkin, 1966). Whether the absence of giant cells
from 19 implants was because they did not develop or were too advanced in
pycnosis to be identified remains conjectural. No qualitative differences were
noticed between implants developed from maximal and equatorial trophoblastic
fragments.
There was no evidence that the trophoblast had proliferated normally and
subsequently dispersed from the implantation chamber. The low number of
trophoblast cells and absence of mitotic figures at all the stages examined leads
to the inescapable conclusion that the trophoblastic fragments did not proliferate
in utero. This might be because the mass of trophoblast tissue, though able to
induce a decidual response, is insufficient to enable subsequent proliferation.
This explanation is ruled out by the fact that partial and half blastocysts containing as few trophoblast cells as trophoblastic fragments can give rise to
normal conceptuses and young at term (Gardner, 1972).
A further possibility might be that the cells overlying the ICM are required
for normal development of the trophoblast. The notion of regional differentiation
of trophoblast is implicit in this explanation. Although the ectoplacental cone
develops over the ICM in the early egg-cylinder (Snell & Stevens, 1966), 'reconstituted' blastocysts specifically lacking embryonic polar trophoblast can
develop normally (Gardner, 1971, and unpublished observations).
The remaining and perhaps most obvious difference between trophoblastic
fragments that fail to produce significant trophoblastic development and intact,
partial or 'reconstituted' blastocysts that do, is that the latter all contain some
ICM tissue. Hence it is concluded that ICM tissue is required to permit normal
proliferation of trophoblast of the 3^-day mouse blastocyst. Abundant trophoblast is found in some implants developed from partial blastocysts that do not
contain embryos (Gardner, 1972), so proliferation presumably does not depend
on development of a definitive embryo. Also, dependence on the ICM is probably limited to an early stage of trophoblast development since ectoplacentacones from 6- to 7^-day egg-cylinders can proliferate following transfer
ectopically (Grobstein, 1950; Billington, 1965; Simmons & Russell, 1966; Clarke,
1969) or to the uteri of cyclic or pseudopregnant mice (Kirby, 1965, 1970;
Kirby & Cowell, 1968).
If the proliferation of trophoblast does indeed depend initially on the ICM
one would expect to find derivatives of the latter tissue in implants with abundant trophoblast. This expectation was fulfilled in nearly all implanted partial and
'reconstituted' blastocysts that did not develop foetuses (Gardner, 1972). The
exceptions may have been more apparent than real since they all showed signs
of cellular degeneration. One of the three atypical implants developing from
304
R. L. GARDNER AND M. H. JOHNSON
implanted trophoblast in the present experiments carried a foetus, and the
second is believed to have acquired its trophoblast from the first. The third
showed a continuous Reichert's membrane enclosing some cells (Fig. 7). This
membrane has been found by elegant immunofluorescence studies to be a secretion of the distal endoderm (Pierce, Midgley, Sri Ram & Feldman, 1962;
Midgley & Pierce, 1963) and hence presumably an ICM derivative (Snell &
Stevens, 1966).
There are few published reports containing detailed descriptions of uterine
implants lacking embryos. Tarkowski (1962) found that rat eggs that had undergone blastulation in the oviduct of the mouse showed dispersal of their ICMs
and developed poorly after implanting in the rat uterus. In extreme cases no
embryo had formed, but profuse trophoblast surrounded a Reichert's membrane
lined with a single layer of cells (Tarkowski, 1962, plate 4, fig. V). The inner
cells are presumably those of the distal endoderm. A similar condition was
found in another implant following transfer of rat eggs to the oviduct of the rat
(Tarkowski, 1962, plate 4, fig. U).
The relevance of descriptions of 20 mouse implantation chambers in which
spontaneous embryonic death had occurred is limited both by the uncertainty
regarding when development went awry and by the fact that the material was
obtained late in pregnancy (Droogleever Fortuyn, 1920). Hence the absence of
all ICM derivatives from five chambers that only contained clusters of giant cells
does not preclude their being formerly present.
Failure of the trophoblast of mouse blastocysts to grow ectopically in recipients specifically immunized against the donor strain while similar ectoplacental
cones succeed has been considered evidence that the former express antigens and
the latter do not (Simmons & Russell, 1966). Present considerations raise the
possibility that it is the ICM of the blastocyst that is susceptible to damage or
immunological attack and that failure of trophoblast development depends
secondarily on this factor (Gardner, Johnson & Edwards, 1972).
Critical appraisal of the relationship between the trophoblast and ICM in
rodent embryos transferred to ectopic sites is complicated by factors such as
abnormal morphogenesis, haemorrhage and degenerative changes. Consequently
many authors fail to specify derivatives other than trophoblast or embryo
proper (e.g. Jollie, 1961). The general conclusion from numerous studies is that
proliferation of the trophoblast is favoured and embryonic development poor
and infrequent, especially if oviducal stages rather than blastocysts are transferred (Billington, Graham & McLaren, 1968; Kirby, 1970). Nevertheless,
Reichert's membrane is found frequently in grafts lacking embryos. Even when
absent as a discrete membrane it may indeed be present as a homogeneous
matrix with cells embedded in it (e.g. Fawcett, 1950, figs. 6 and 7; Whitten, 1958,
fig. 2; McLaren & Tarkowski, 1963, fig. 4; and author's unpublished observations). Such 'abortive yolk sacs' closely resemble the undifferendated
murine teratocarcinoma embedded in neoplastic hyalin with which they
Inner cell mass and twphoblast tissues
305
are almost certainly homologous (Pierce et al. 1962; Midgley & Pierce,
1963).
A particularly illuminating study, in which all host kidneys were examined
after transfer of 3^-day mouse blastocysts, is reported by Johnson in an appendix
to this paper. Only two grafts definitely lacked ICM derivatives and these, like
most uterine implants developed from trophoblastic fragments, contained just
a few giant cells. Such grafts are likely to have been overlooked in studies where
host organs showing macroscopic 'takes' were selected for examination.
Any explanation of the relationship deduced between trophoblast and ICM
must account for the failure of proliferation of implanted trophoblastic fragments
on the one hand, but development of some giant cells in most of them on the
other. Available data are embraced by the following hypothesis. The mural
trophoblast of the mouse blastocyst which gives rise to the primary giant cells
(Dickson, 1963) can do so autonomously, or has already received the appropriate
stimulus before it is separated from the ICM. It thus provides the few giant cells
found in trophoblastic implants. The development of the ectoplacental cone
(and hence the multitude of secondary giant cells derived from it, Snell &
Stevens, 1966) is specifically dependent on the presence of ICM tissue in the
blastocyst or early egg-cylinder. The role of the ICM might be to promote
division or inhibit giant transformation of the overlying trophoblast cells. The
notion of an inductive interaction is attractive because the ectoplacental cone
invariably develops over the ICM, and because the latter may attain its final
mesometrial position by migrating round the inner surface of the trophoblast
wall (Kirby, Potts & Wilson, 1967; Jenkinson & Wilson, 1970; Gardner, 1971).
However, it is also possible that the ectoplacental cone is a derivative of the
ICM (Duval, 1892).
Finally, the morphological similarity between early implants of pure trophoblast and those of homozygous lethal yellow embryos in utero has not escaped
notice (fig. 9 A, B; Eaton & Green, 1963). It is of considerable interest to discover
whether this resemblance is more than superficial.
I wish to thank Professor C. R. Austin, Dr R. G. Edwards, Dr C. F. Graham, Dr M. H.
Johnson and Dr M. I. Sherman for advice and discussion, and Mrs S. C. Barton, Mrs W. J.
Gardner and Mrs W. Redmond for help. This work was supported by the Medical Research
Council, the World Health Organisation and the Ford Foundation.
306
R. L. GARDNER AND M. H. JOHNSON
APPENDIX
Relationship between inner cell mass derivatives and
trophoblast proliferation in ectopic pregnancy
SUMMARY
Forty-two 3^-day mouse embryos were transferred to ectopic sites in the kidneys of male
mice. Microscopic investigation of all transfers revealed that trophoblast proliferation had
occurred only when inner cell mass derivatives were present. In the absence of inner cell mass
derivatives, non-proliferated giant cells were present. This data is taken as evidence compatible
with the hypothesis that the inner cell mass is essential for trophoblast proliferation.
The results achieved by the use of elegant microsurgical techniques have led
Gardner to formulate the hypothesis that the inner cell mass of the 3^-day
mouse embryo is essential for development of normal proliferated trophoblast
(see the first part of this paper). In the presence of the inner cell mass, normal
ectoplacental cone tissue with its peripheral secondary giant cells develops. In
the absence of an inner cell mass, the mural trophoblast of the 3^-day blastocyst
merely differentiates into primary giant cells without prior division.
This hypothesis should also apply to ectopically transferred 3^-day embryos,
but appears at first sight to be refuted by the reports emphasizing substantial
trophoblast proliferation with little or no embryonic development in such
conditions (Fawcett et al. 1947; Runner, 1947; Fawcett, 1950; Kirby, 1960,
1962). In fact, careful examination of these reports in most cases reveals a description of some derivatives of inner cell mass such as an abortive yolk sac
(Fawcett, 1950) or some more complex, but imperfect structure of proven inner
cell mass origin (Snell & Stevens, 1966). With the hypothesis of Gardner in mind,
careful histological analysis was made of 42 mouse embryos (3^-day, C3H or
C 5 7 B L ) transplanted ectopically to the kidneys of male mice of homologous
strains. The transfer technique has been described elsewhere (Johnson &
Dharmawardena, 1972). Seven days after transfer, all kidneys were removed,
fixed and serial sections were cut and examined regardless of whether the embryo
appeared to have 'taken' macroscopically. Each embryo found was classified
into one of the five categories described in Table 7. The classification of group 4
transplants (Fig. 13) as Reichert's membrane plus endoderm-like nuclei was made
by others (Fawcett, 1950; Stevens, 1968) and has been discussed in detail by
Gardner in the first part of this paper.
The data show that, with one exception, trophoblastic proliferation and the
presence of secondary type giant cells were detected only where some evidence
of inner cell mass derivatives existed. For the exceptional transplant, an area of
necrosis was present in the core of the ectoplacental cone tissue normally occupied
by the embryo. For two transplants, in which inner cell mass derivatives were
not present, small nodules of primary giant cells were detected (Fig. 14). No
Inner cell mass and trophoblast tissues
307
Table 7
Classification of transplant
No. of
embryos
1. Embryo showing normal organization at centre
of proliferating ectoplacental cone with
numerous peripheral secondary-type giant cells.
2. Embryo showing minor abnormalities or areas
of necrosis at centre of proliferating ectoplacental
cone with numerous peripheral secondary-type
giant cells.
3. Area of necrotic cells (probably embryo)
surrounded by proliferating ectoplacental cone
with numerous peripheral secondary-type giant
cells.
4. Compact nodule of Reichert's membrane and
distal endoderm surrounded by proliferating ectoplacental cone with numerous peripheral secondarytype giant cells (Fig. 13).
5. A few non-proliferated, primary-type giant cells
only. No proliferating ectoplacental cone or inner
cell mass derivatives (Fig. 14).
6
10
1
23
2
proliferating trophoblast of the type seen in the ectoplacental cone of uterine
or other ectopic transplants was present, suggesting that the giant cells were
primary rather than secondary. The number of cells in each nodule, 9 and 21,
were of the order expected for primary trophoblast after transfer of the 30-40
mural trophoblast cells in the 3^-day embryo (Gardner, above). The giant cells
were not surrounded by any extensive necrotic or scar tissue, thereby negating
the possibility that they were the residuum of a proliferated transplant that
subsequently died. In fact, the two clusters of giant cells bore a remarkable
resemblance, both histological and in terms of cell numbers, to those occurring
in utero after transfer of pure trophoblastic vesicles (Gardner, above). Presumably the inner cell masses of these two embryos had been damaged on transfer,
resulting in the absence both of inner cell mass derivatives, and, according to the
hypothesis of Gardner, of a stimulus to the proliferation of ectoplacental
trophoblast. The trophectoderm of the transplanted blastocyst therefore merely
differentiated into primary giant cells without prior division.
Examination of the literature on ectopic transfers has revealed only one study
in which ectopic transfer sites have been examined histologically regardless of
whether a macroscopic haemorrhage was present (Stevens, 1968). In that study
also, non-proliferated primary trophoblastic giant cells only evidently occurred
in the absence of any inner cell mass derivatives. Careful analysis of ectopic
transfers in other studies would be worth while. The absence of such analyses in
308
R. L. GARDNER AND M. H. JOHNSON
Fig. 13. Nodule of Reichert's membrane and distal endoderm nuclei embedded
in proliferating trophoblast. x 720.
ectopic transfers of blastocysts to recipients preimmunized against donor histocompatibility antigens may have led to misleading conclusions about the distribution of these antigens on the early embryo. The failure to detect macroscopic
haemorrhage in these studies could have been due to failure of trophoblast to
proliferate secondary to immunological destruction of the inner cell mass rather
Inner cell mass and trophoblast tissues
309
Fig. 14. Non-proliferated trophoblastic giant cells embedded in kidney tissue.
xl80.
than to a primary immunological destruction of the trophoblast (Gardner et
al. 1972).
In conclusion, the findings that trophoblastic proliferation is invariably
associated with the presence of inner cell mass derivatives and that a failure of
trophoblastic proliferation is associated with the absence of inner cell mass
310
R. L. GARDNER AND M. H. JOHNSON
derivatives are not in conflict with the hypothesis of Gardner (above) and indeed
offer positive support for it.
I wish to acknowledge the stimulation provided by discussion with Richard Gardner and
the technical assistance of Vinitha Dharmawardena. The work was done whilst the author was
in receipt of an M.R.C. Junior Fellowship, and was supported by a grant to Professor C. R.
Austin from the Ford Foundation.
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(Manuscript received 22 March 1972)