/. Embryol. exp. Morph. Vol. 36, 1, pp. 163-174, 1976
Printed in Great Britain
\ 63
Investigation of inner cell mass
determination by aggregation of isolated rat inner
cell masses with mouse morulae
By J. ROSSANT 1
From the Department of Zoology, Oxford
SUMMARY
Inner cell masses (ICMs) dissected from 4-^-day rat blastocysts were aggregated with 2^-day
mouse morulae. Successful aggregates formed blastocysts in vitro and morphologically normal
5i-day conceptuses in the mouse uterus. Immunofluorescent analysis of these conceptuses
revealed that rat cells were only present in the embryonic ectoderm and endoderm and never
in the trophectoderm derivatives, although rat trophoblast did develop in the mouse uterus in
various control experiments. The single-cell resolution of this technique extends the results
obtained from aggregating mouse ICMs with mouse morulae and provides strong evidence
that ICM cells, although not overtly differentiated, are determined by the blastocyst stage.
INTRODUCTION
The inner cell mass (ICM) isolated from the mouse blastocyst, although not
an overtly differentiated tissue (Gardner, 1972) is apparently determined by
3% days post coitum, as judged by its inability to contribute to post-implantation
trophoblast after aggregation with morulae or after transfer to the oviduct and
later injection into blastocysts (Rossant, 1975 a, b). In these experiments, electrophorettc variants of glucose phosphate isomerase (GPI) were used as genetic
markers. However, the low sensitivity of this technique means that a minor
contribution of ICM cells to the proliferating trophoblast or a contribution to
the non-dividing mural trophoblast giant cells cannot be excluded (Gardner,
Papaioannou & Barton, 1973). A marker that can detect individual cells is required to eliminate this possibility. No such markers are yet available for the
mouse (Gardner & Johnson, 1975), but immunofluorescent analysis of rat/
mouse chimaeras permits identification of single rat or mouse cells (Gardner
& Johnson, 1973, 1975). Thus, an attempt was made to extend the results of
experiments using mouse ICM/mouse morula aggregates by aggregating rat
ICMs with mouse morulae. Immunofluorescent analysis can then be used to
discover whether any rat ICM cells contribute to the trophoblast of resulting
conceptuses. Analysis was performed at 5^ days of pregnancy in the mouse
1
Author's address: Department of Zoology, South Parks Rd., Oxford 0X1 3PS, U.K.
II-2
164
J. ROSSANT
when all three presumptive derivatives of the trophectoderm - ectoplacental
cone, primary giant cells and extra-embryonic ectoderm - should be present
(Gardner & Johnson, 1975; Gardner & Papaioannou, 1975).
MATERIALS AND METHODS
Recovery of embryos from donor females
Mouse morulae (8-16 cell) were flushed from the oviducts of CFLP females
(Anglia Laboratory Animals Ltd) on the afternoon of the 3rd day after mating.
Rat morulae at similar cleavage stages were flushed from the uterotubal junctions of CFHB random-bred females (Anglia Laboratory Animals Ltd) on the
afternoon of the 4th day after mating, while rat blastocysts were obtained from
the uterus on the afternoon of the 5th day. It is assumed throughout that rat
early development is 24 h behind that of the mouse.
PB1 medium +10 % foetal calf serum (FCS) was used for recovery, storage,
microsurgery and transfer of embryos (Whittingham & Wales, 1969) and Ml6
medium + 10 % FCS was used for culture (Whittingham, 1971).
Aggregation of culture and embryos
1. Rat ICM/mouse morula aggregates
ICMs were dissected from 4^-day rat blastocysts and aggregated with mouse
morulae as described previously for mouse ICM/mouse morula aggregates
(Rossant, 1975a). All aggregates were cultured in M16. A few pairs were cultured to the late blastocyst stage to observe and photograph the process of
aggregation. The remainder were cultured for either 2-4 h or 24 h (late
morula/early blastocyst) before transfer to pseudopregnant mice.
2. Rat blastocysts
Some 4^-day rat blastocysts were not dissected but were stored briefly in PB1
+10 % FCS before transfer to mice.
3. Rat morulae
Rat morulae (3£ days) were cultured for 24 h in M16 or M16 +10 % FCS
before transfer to recipient rats or mice. In most cases the zonae were removed
by Pronase (Mintz, 1962) before culture.
4. Rat morula /mouse morula aggregates
The zonae were removed from both rat and mouse morulae by Pronase and
rat/mouse pairs were brought into contact in drops of M16 or M16 +10 % FCS.
The cultures were then transferred to a 37 °C incubator. Contact between the
morulae was checked after 1 h. After 24 h in culture, aggregated embryos were
transferred to pseudopregnant mice.
Investigation of inner cell mass determination
165
Transfer to recipient animals
All four types of embryos listed above were transferred to recipient mice on the
3rd day of pseudopregnancy. Rat recipients were used for uterine transfer on the
4th day of pregnancy after the oviducts had been ligated on day 2. Rat embryos
cultured for 24 h were transferred unilaterally so that the contralateral horn
provided a control for effective ligation of the oviduct.
Analysis of post-implantation development
Histology
Recipient mice which had received non-cultured or cultured rat embryos, and
one recipient which contained rat morula/mouse morula aggregates, were killed on
the 6th day after mating (5^-day embryos). Uteri containing implants were fixed
in AFA, processed and embedded in wax (Orsini, 1962). Serial sections were cut
at 6-7 /.im, stained with haemalum and eosin and examined for embryonic
derivatives.
Recipient rats which had received cultured rat embryos were killed on the
7th day after mating and uteri containing implants were processed as above.
Immunofluorescent analysis
Mice which had received rat ICM/mouse morula aggregates and rat morula/
mouse morula aggregates were killed on the sixth day of pregnancy. Decidua
were processed and embedded in wax (Sainte-Marie, 1962) and sectioned at
6 jam. The sections were treated with antisera specific for mouse and rat antigens as described previously (Gardner & Johnson, 1973) and examined by
epifluorescent illumination in a Zeiss fluorescence microscope (Primary filter,
barrier filter HBO 200). Camera lucida drawings were made of all sections,
marking rat and mouse cells.
RESULTS
In vitro observations
1. Rat ICMjmouse morula aggregates
Successful aggregation of rat ICMs with mouse morulae was achieved in vitro
in Ml6 (Table 1). The rate of aggregation was lower than for mouse ICM/
morula combinations (Rossant, 1975 a) but a large proportion of successful
aggregates formed blastocysts after 24 h in culture (Table 1).
2. Rat morulae
Rat morulae were cultured to the blastocyst stage in both M16 and M16 +
10 % FCS. However, success was rather variable and only 32/66 and 10/28
morulae respectively underwent further cleavage.
166
J. ROSSANT
Table 1. Rates of in vitro aggregation and blastocyst formation of rat
ICMj mouse morula and rat morula I mouse morula combinations
Type of combination
Rat ICM/mouse
morula
Rat morula/mouse
morula
Rat morula/mouse
morula
Culture medium No. pairs
No. aggregated
(percentage
aggregation)
No. blastocysts
formed (percentage formation)
M16
48
23(48)
19(83)
Ml6
45
31(69)
26(84)
M16 +10 % FCS
143
65(45)
45(69)
Table 2. Implantation rates {only pregnant recipients considered)
Type of embryo transferred
Rat ICM/mouse morula
aggregates
Rat blastocysts
Cultured rat blastocysts
Cultured rat blastocysts
Rat morula/mouse
morula aggregates
Type of
recipient
No.
No.
recipients transferred
No. (%)
implants
No. (%)
embryos
Mouse
5
22
11(50)
7(32)
Mouse
Mouse
Rat
Mouse
2
4
4
8
10
16
22
36
9(90)
10(62)
8(36)
23C64)
8(80)
8(50)
4(18)
14(39)
3. Rat morulajmouse morula aggregates
Successful aggregation of rat and mouse morulae was achieved in culture in
both M16 and M16 +10 % FCS (Table 1). The rather low rate of aggregation
was chiefly due to the fairly frequent failure of rat morulae to develop in culture,
as reported above. A large proportion of the successful aggregates formed blastocysts after about 24 h in culture (Table 1), as reported previously by other
workers (Mulnard, 1973; Stern, 1973; Zeilmaker, 1973).
Analysis of post-implantation development
1. Rat ICMjmouse morula aggregates
The implantation rate of these aggregates was rather low (Table 2; see
Rossant, 1975 a). However, seven embryos were obtained and examined by
immunofluorescence. Five proved to be interspecific chimaeras; their patterns
of chimaerism are summarized in Table 3. Conceptus 90a was obtained from an
aggregate transferred after 2-4 h in culture, whereas the others were cultured
for 24 h before transfer. All five were morphologically normal although embryos
90a and 95b were retarded slightly and lacked extra-embryonic ectoderm. None
showed contribution of rat cells to the mural trophoblast, ectoplacental cone or
extra-embryonic ectoderm.
Investigation of inner cell mass determination
167
Table 3. Estimation by immunofluorescence of the proportion of rat cells in
5\-day conceptuses derived from rat ICMjmouse morula aggregates
Conceptus
code no.
89a
89c
89e
90a
95b
Total + for
each tissue
Total no.
embryos with
rat cells in
given tissue
Mural Polar
tropho- trophoblast
blast
Proximal
endoderm
Distal
endoderm
ExtraEmbryonic embryonic
ectoderm ectoderm
—
—
++
++
+++
—
—
—
—
—
—
—
—
—
+
—
++++
—
+
++
+++
—
—
—
++
++++
—
—
n.p.
n.p.
0
0
7
8
9
0
0
0
3
4
3
0
Key to tables 3 and 4. + signs represent visual estimations of the proportion of rat cells
in each tissue. + + + + = all rat; + + + = mostly rat > 50%; + + = some rat < 50%;
+ = very little rat; - = no rat; n.p. = tissue not present.
2. Rat blastocysts
As a control for survival of rat trophoblast in the mouse uterus, non-cultured
rat blastocysts were transferred to recipient mice. A high proportion were
capable of inducing decidual formation (Table 2). Histological examination of
the 5-|-day decidua revealed that most contained embryonic cells. Six out of
eight contained a definite egg-cylinder but this was never well organized (Fig. 1).
Ectoderm and endoderm were generally delineated but no division into embryonic and extra-embryonic ectoderm was apparent and the orientation of
the embryos was often abnormal. All conceptuses contained trophoblast cells
and in six out of eight definite mural giant cells could be identified (Fig. 1).
Pycnotic cells were present to a greater or lesser extent in all conceptuses.
3. Rat morulae
To control for the ability of rat trophoblast to survive in the mouse after
development in culture, blastocysts developed from rat morulae cultured for
24 h in M16 + 10%FCS were transferred to pseudopregnant mice. Decidual
formation was induced (Table 2) and 50 % of the embryos transferred produced
embryonic structures. Four such structures consisted solely of groups of trophoblast cells with some giant cell formation (Fig. 2). The other four consisted of
well-expanded trophoblastic vesicles, containing only a few dispersed cells on
their inner surface (Fig. 3). No organized ICM was present and the inner cells
were presumably endoderm. The beginning of giant cell formation was also
apparent in these structures.
168
J. ROSSANT
In all figures, the grid bar represents 50/<m.
Fig. 1. Section of 5^-day conceptus developed from non-cultured rat blastocyst
transferred to the mouse uterus. Arrows indicate definite trophoblast giant cells.
When rat blastocysts cultured in Ml6 alone were transferred to rats, the implantation rate was low (Table 2). However, considering the fairly high implantation rate in transfers to mice, this may be largely due to lack of practice in
performing rat uterine transfers. Of the eight decidua formed, four had normal
rat egg-cylinders and two had trophoblast cells only.
Investigation of inner cell mass determination
169
Fig. 2. Group of trophoblast cells produced 5-j- days after transfer of cultured
rat blastocyst to the mouse uterus. Arrows indicate definite trophoblast giant cells.
4. Rat morulajmouse morula aggregates
To control for the development of rat trophoblast in competition with mouse
cells, rat morula/mouse morula aggregates were transferred to mouse uteri. The
number of post-implantation embryos formed from these aggregates was rather
170
J. ROSSANT
Fig. 3. Section of 5^-day trophoblast vesicle containing only a few endoderm cells
after transfer of cultured rat blastocyst to the mouse uterus.
low (Table 2). The first three embryos were examined histologically and were
found to be egg-cylinder-like but not well organized (Fig. 4). Trophoblast,
ectoderm and endoderm were present in all three, but the expected division
into embryonic and extra-embryonic ectoderm was not found. However, since
trophoblast cells were found, a further 11 conceptuses were analysed by immunofluorescence to see if any of the trophoblast cells were rat.
Investigation of inner cell mass determination
171
Fig. 4. Section of 5-i-day conceptus derived from rat morula/mouse morula aggregate.
The patterns of chimaerism in these embryos are summarized in Table 4.
None was a completely normal 5-|-day embryo. Some resembled retarded blastocysts (e.g. R/M 19.3-4) while others were more advanced but more disorganized
(e.g. R/M 19.3-3). The orientation of some was abnormal (e.g. R/M 28.3-2).
However, distal and proximal endoderm, mural trophoblast, ectoplacental cone
and embryonic ectoderm were present in all embryos. There was a definite
tendency for the rat cells to colonize the endoderm in preference to other tissues
(Table 4) but 7 out of 11 contained rat ectoderm cells. Also, 10 out of 11
contained rat giant trophoblast cells, and 9 out of 11 rat polar trophoblast.
172
J. ROSSANT
Table 4. Estimation by immunoftuorescence of the proportion of rat cells in
5\-day conceptuses derived from rat morulajmouse morula aggregates
Conceptus
code no.
Mural
trophoblast
92a74
+
96b74
—
R/M 19-3-1
+
R/M 19-3-2
++
R/M 19-3-3
++
R/M 19-3-4
++
R/M 28-3-1
+++
R/M 28-3-2
+
R/M 28-3-3
++
R/M 9-5
+
R/M 15-5
++
Total + for each tissue 17
Total no. embryos
with rat cells in
given tissue
10
Polar
trophoblast
Proximal
endoderm
+
—
—
++
++
+++
++
++
+
+
+++
17
++
++
+++
+++
++
++
++++
+++
+++
+++
+++
30
9
11
Distal
endoderm
Embryonic
ectoderm
+++
++
++
—
++
—
++
—
+++
++
+
—
+++
++
++
++
+++
+
++++ +
+++
++
++
27
15
11
7
Conceptuses 92a74 and 96b74 and the three conceptuses examined histologically were derived from aggregates cultured in M16 while the rest had been
cultured in M16 + 10%FCS. No obvious differences in morphology were
apparent between the two groups of embryos.
DISCUSSION
Aggregation of 4^-day rat ICMs with 2^-day mouse morulae has been achieved
in vitro and morphologically normal blastocysts were produced. After transfer
to pseudopregnant mice, these blastocysts formed apparently normal 5^-day
conceptuses, of which a high proportion (5/7) were inter-specific chimaeras.
However, immunofluorescent analysis revealed rat cells only in the presumptive
ICM derivatives, i.e. embryonic ectoderm, distal and proximal endoderm and
never in the trophectoderm derivatives, i.e. ectoplacental cone, extra-embryonic
ectoderm and mural trophoblast (Gardner & Johnson, 1975; Gardner & Papaioannou, 1975). Because of the single-cell resolution of the analysis these results
completely exclude the possibility of a contribution, however minor, of ICM
cells to the trophoblast. Thus they extend the conclusions drawn from GPI
analysis of mouse ICM/mouse morula aggregates reported previously (Rossant,
1975 a).
However, since interspecific chimaeras were used, the validity of the present
results could be queried, unless it can be shown that rat trophoblast can develop
in the mouse until at least 5^ days of pregnancy. Non-cultured rat blastocysts produced both proliferating trophoblast and giant cells at 5£ days in the
mouse, although the embryonic regions were rather disorganized (Fig. 1).
Investigation of inner cell mass determination
173
Culture of rat morulae to the blastocyst stage before transfer to mice also did
not prevent trophoblast formation although no conceptus contained an organized ICM (Figs. 2, 3). Finally, rat trophoblast, both proliferating and giant,
could even be formed in competition with mouse cells in rat morula/mouse
morula aggregates (Table 4, Fig. 4). Development of rat trophoblast in the
mouse under such a variety of conditions means that the absence of rat
trophoblast in conceptuses derived from rat ICM/morula aggregates can be
considered as a valid result. Thus, the present experiments provide strong
evidence that ICM cells, although not overtly differentiated, are determined by
the blastocyst stage. This is supported by recent evidence of molecular differentiation between ICM and trophectoderm (Van Blerkom, Barton & Johnson,
1976).
The present experiments also provide some new information on the morphology of rat/mouse chimaeras. Chimaeras derived from rat ICM/mouse morula
aggregates are morphologically normal at least in early stages, as are those
produced by injection of rat ICMs into mouse blastocysts (Gardner & Johnson,
1973, 1975). However, none of the 14 5^-day conceptuses derived from rat
morula/mouse morula aggregates could be considered normal (Fig. 4). The
reasons for this difference are not clear, but at least two possible explanations
can be suggested.
Firstly, culture may produce deleterious effects on rat morulae and not on rat
ICMs, since consistent success in culturing rat morulae to the blastocyst stage
was not achieved. In particular, the predominance of endodermal chimaerism in
rat morula/mouse morula aggregates (Table 4) might be related to the effects of
culture. Cultured rat blastocysts never produced egg-cylinder structures when
transferred to the mouse, but four out of eight produced trophoblast vesicles
containing only a few dispersed endoderm cells. However, six out of eight noncultured rat blastocysts did produce egg-cylinders containing ectoderm cells.
Thus there seems to be a correlation between culture of rat embryos and lack of
ectoderm formation, although Tarkowski has observed similar trophoblast
vesicles containing only endoderm cells after transfer of rat eggs to the mouse
oviduct (Tarkowski, 1962).
The second factor exerting a deleterious effect on the development of rat
morula/mouse morula chimaeras may be the formation and continued presence
of rat trophoblast, since neither rat ICM/mouse morula nor rat ICM/mouse
blastocyst chimaeras contain this tissue. Tarkowski (1962) has suggested that
the abnormal development of rat eggs after implantation in the mouse may be
due to defective interaction of the rat trophoblast with the mouse uterine epithelium. Similar effects could be occurring in some rat morula/mouse morula
aggregates. Mystkowska (1975) has reported similar poor postimplantation
development from bank vole morula/mouse morula aggregates, and she suggested that the wide taxonomic gap and differences in the course of embryogenesis between the two species were the main causes. However, the present
174
J. ROSSANT
experiments indicate that even morulae of more closely related species like the
mouse and the rat are rarely able to integrate and form normal conceptuses.
I should like to thank Dr R. L. Gardner, Dr M. H. Johnson and Mr A. Copp for valuable
discussion. Special thanks are due to Dr Johnson, who provided the specific antisera and
laboratory facilities for performing the immunofluorescent analysis. Mrs L. Ofer and Mrs
S. Clutterbuck-Jackson provided technical assistance. The author was supported by a Medical
Research Council Research Studentship and a Beit Memorial Junior Research Fellowship.
The work was supported by Medical Research Council project grants to Dr Gardner and
Dr Johnson.
REFERENCES
R. L. (1972). An investigation of inner cell mass and trophoblast tissue following
their isolation from the mouse blastocyst. /. Embryol. exp. Morph. 28, 279-312.
GARDNER, R. L. & JOHNSON, M. H. (1973). Investigation of early mammalian development
using interspecific chimaeras between rat and mouse. Nature (New BioL), Lond. 246, 86-89.
GARDNER, R. L. & JOHNSON, M. H. (1975). Investigation of cellular interaction and deployment in the early mammalian embryo using interspecific chimaeras between the rat and
mouse. In Cell Patterning (Ciba Found. Symp. 29), pp. 183-200. Amsterdam: Associated
Scientific Publishers.
GARDNER, R. L. & PAPAIOANNOU, V. E. (1975). Differentiation in the trophectoderm and
inner cell mass. In The Early Development of Mammals {2nd Symposium of the British
Society for Developmental Biology) (ed. M. Balls & A. E. Wild), pp. 107-132. Cambridge
University Press.
GARDNER, R. L., PAPAIOANNOU, V. E. & BARTON, S. C. (1973). Origin of the ectoplacental
cone and secondary giant cells in mouse blastocysts reconstituted from isolated trophoblast and inner cell mass. /. Embryol. exp. Morph. 30, 561-572.
MINTZ, B. (1962). Experimental study of the developing mammalian egg: removal of the zona
pellucida. Science, N. Y. 138, 594-595.
MULNARD, J. G. (1973). Formation de blastocystes chimeriques par fusion d'embryons de rat
et de souris au stade VIII. C. r. hebd. Seanc. Acad. Sci. Paris 276, 379.
MYSTKOWSKA, E. T. (1975). Development of mouse-bank vole interspecific chimaeric embryos.
/. Embryol. exp. Morph. 33, 731-744.
ORSINI, M. W. (1962). Technique of preparation, study and photography of benzyl-benzoate
cleared material for embryological studies. /. Reprod. Fert. 3, 283-287.
ROSSANT, J. (1975a). Investigation of the determinative state of the mouse inner cell mass.
I. Aggregation of isolated inner cell masses with morulae. /. Embryol. exp. Morph. 33,
979-990.
ROSSANT, J. (19756). Investigation of the determinative state of the mouse inner cell mass.
II. The fate of isolated inner cell masses transferred to the oviduct. /. Embryol. exp. Morph,
33,991-1001.
SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10, 250-256.
STERN, M. S. (1973). Chimaeras obtained by aggregation of mouse eggs with rat eggs. Nature,
Lond. 243, 472-473.
TARKOWSKI, A. K. (1962). Interspecific transfers of eggs between rat and mouse. /. Embryol.
exp. Morph. 10, 476^95.
VAN BLERKOM, J., BARTON, S. C. & JOHNSON, M. H. (1976). Molecular differentiation in the
preimplantation mouse embryo. Nature, Lond. 259, 319-321.
WHITTINGHAM, D. G. (1971). Culture of mouse ova. /. Reprod. Fert. Suppl. 14, 7-2.1.
WHITTINGHAM, D. G. & WALES, R. G. (1969). Storage of two-cell mouse embryos in vitro.
Aust. J. BioL Sci. 22, 1065-1068.
ZEILMAKER, G. H. (1973). Fusion of rat and mouse morulae and formation of chimaeric
blastocysts. Nature, Lond. 242, 115-116.
GARDNER,
(Received 28 January 1976; revised 26 April 1976)