/ . Embryol. exp. Morph. Vol. 39, pp. 183-194, 1977
Printed in Great Britain
183
Properties of extra-embryonic ectoderm isolated
from postimplantation mouse embryos
By J. ROSSANT AND L. OFER 1
From the Department of Zoology, Oxford
SUMMARY
Extra-embryonic ectoderm isolated from the mouse embryo as late as 8i days post coitum
can form cells with the morphological characteristics of trophoblast giant cells both in ectopic
sites and in vitro. This similarity to the properties of ectoplacental cone tissue provides further
support for the postulated common origin of both tissues from the trophectoderm of the
blastocyst.
INTRODUCTION
Embryologists have disagreed over whether the extra-embryonic ectoderm
of the mouse egg cylinder arises from the trophectoderm (Jenkinson, 1900)
or the inner cell mass (ICM) (Robinson, 1904) of the blastocyst. The extraembryonic ectoderm lies between the ectoplacental cone and the embryonic
ectoderm in the early egg cylinder. It later forms the ectoderm of the chorion
which fuses with the ectoplacental cone to produce the trophoblastic layers of
the placenta (Duval, 1891; Jenkinson, 1902). If the extra-embryonic ectoderm
is derived from the trophectoderm of the blastocyst, this would suggest a
unitary origin of all trophoblast tissues, since the trophectoderm is already
known to give rise to the ectoplacental cone (Gardner, Papaioannou & Barton,
1973). Recent experiments tend to support this hypothesis. 'Reconstituted
blastocyst' experiments revealed a trophectoderm contribution to the 'embryo
plus membranes' fraction of later conceptuses (Gardner et al. 1973). Injection of
rat ICMs into mouse blastocysts suggested that this contribution was to the extraembryonic ectoderm, since no rat cells were ever found in the extra-embryonic
ectoderm of resulting interspecific chimaeras, even where all the embryonic
ectoderm was of rat origin (Gardner & Johnson, 1973, 1975).
On the basis of this evidence, Gardner & Papaioannou (1975) suggested
that the trophectoderm cells over the ICM of the blastocyst proliferate and
push inwards to form the extra-embryonic ectoderm as well as outwards to
form the ectoplacental cone (see figure 1, Gardner & Papaioannou, 1975). If
this interpretation is correct, the distinction usually drawn between the ectoplacental cone and the extra-embryonic ectoderm at the origin of Reichert's
membrane (Snell & Stevens, 1966, figure 12.8) is purely arbitrary and the two
1
Authors' address: Department of Zoology, South Parks Road, Oxford 0X1 3PS, U.K.
184
J. ROSSANT AND L. OFER
tissues should have similar properties. Preliminary experiments suggest that
this is so. 5^-day extra-embryonic ectoderm grafted under the testis capsule
produced haemorrhagic grafts containing giant cells similar to those produced
by grafted ectoplacental cones (Gardner & Papaioannou, 1975). Diwan &
Stevens (1975) report similar results with 6-day extra-embryonic ectoderm.
Extra-embryonic ectoderm isolated from embryos on the 6th and 7th days of
pregnancy also formed cells morphologically similar to trophoblast giant cells
when grown in vitro (Gardner & Ofer, unpublished results).
The aim of the present experiments was to extend these preliminary studies
to discover how late in development isolated extra-embryonic ectoderm retains
the capacity to form giant cells. The isolated tissues were either transferred to
ectopic sites, or cultured in vitro so that the cells could be readily harvested for
mitotic counts and microdensitometry measurements.
MATERIALS AND METHODS
Recovery of embryos and separation of isolated germ layers
Mice from random-bred Swiss PO stock (Pathology, Oxford) were used
throughout this study. PB1 medium (Whittingham & Wales, 1969) containing
10 % foetal calf serum was used for recovery, storage, dissection and transfer of
embryos. Embryos were dissected from the uteri of mice on the 6th, 7th, 8th
and 9th day of pregnancy (5^-, 6^-, 1\-, 8^-day embryos). Both 5^- and 6^-day
embryos consist of egg cylinders with no mesoderm formation (Snell & Stevens,
1966, figure 12.8). In 7^-day embryos, mesoderm is being formed by the primitive streak and the amniotic folds begin to separate the extra-embryonic ectoderm from the embryonic shield (Snell & Stevens, 1966, figure 12.13). From this
stage onwards the extra-embryonic ectoderm forms the ectoderm of the chorion.
By the next day the amnion is complete and the chorion is flattened against the
ectoplacenta, constricting the ectoplacental cavity. The allantois is also developing but has not yet fused with the chorion (Snell & Stevens, 1966, figure
12.17). Somite formation has begun. After %\ days, clean separation of the
chorionic ectoderm from the allantois and the ectoplacental cone is not
readily achieved.
Reichert's membrane was torn away from the embryos after they were
cleared of uterine tissue and the embryos were cut by hand using glass microneedles. At 5^ and 6^ days, embryos were divided into ectoplacental (very small
and easily damaged at 5% days), extra-embryonic and embryonic regions. Since
no obvious division between the ectoplacental cone and the extra-embryonic
ectoderm was apparent, an arbitrary cut was made below the point of insertion
of Reichert's membrane. At 1\ and 8-^- days, the same divisions were made,
but an extra region including the amniotic folds was removed from the middle
of the egg cylinder (the exocoelomic region) and discarded.
The embryonic and extra-embryonic regions from each embryo were then
Properties of mouse extra-embryonic ectoderm
185
placed in a solution of 2-5 % Pancreatin and 0-5 % trypsin in calcium, magnesium-free Tyrode's saline at 4 °C for 10-20 min. Incubation in this enzyme
solution has been shown to facilitate separation of the germ layers in rat egg
cylinders (Levak-Svajger, Svajger & Skreb, 1969). Separation of the germ layers
in the present experiments was achieved by sucking the embryonic or extraembryonic fragments up and down in flame-polished micropipettes of slightly
narrower diameter than that of the fragments. At 5^ and 6^ days, enzymic
treatment removed endoderm from the outside of the extra-embryonic ectoderm.
At 1\ days, endoderm and extra-embryonic mesoderm came free from the
chorionic ectoderm, and endoderm and some of the mesoderm were separated
from the embryonic ectoderm. At 8^ days, endoderm and mesoderm were again
removed from the chorionic ectoderm but no attempt at germ layer separation
of the complex embryonic region was made.
The results of this combined microsurgical and enzymic treatment was to
produce clean fragments of extra-embryonic and embryonic ectoderm, with
or without some attached mesoderm, from all stages. Ectoplacental cones were
not subjected to enzyme treatment.
Ectopic transfers
Single embryonic ectoderm, extra-embryonic ectoderm and ectoplacental
cone fragments were transferred beneath the testis capsule of male mice using a
micropipette. At 8^ days, the fragments from a single embryo were cut into
smaller pieces before transfer. After 7 days, the recipients were killed and their
testes examined for the presence of haemorrhagic graft sites. All testes, whether
or not showing macroscopic signs of graft survival, were fixed, embedded and
sectioned at 7-8 /«n. The sections were stained with haemalum and eosin and
scanned for the presence of graft derivatives.
In vitro culture
Embryonic ectoderm, extra-embryonic ectoderm and ectoplacental cone
fragments prepared as above were cultured in separate wells of Falcon Microtest II tissue plates at 37 °C. RPMI medium (Flow Labs)+ 10% foetal calf
serum + 2 % glutamine, gassed with 5 % CO2, 90 % N 2 , 5 % O2, was used for
culture and all fragments were grown on a feeder layer of Mitomycin C-treated
mouse fibroblasts (Sto cells) at a concentration of 4 x 104 cells/well. A feeder
cell layer was found to promote growth of the embryonic fragments and so was
used for all explants to standardize culture conditions. The medium was changed
every 2 days for 1 week. After this time, the cell morphology of the explants
was recorded in the inverted phase microscope and some representative explants
were prepared for microdensitometry.
The mitotic activity of 5^- to 8^-day extra-embryonic ectoderm and 1\- and
8^-day embryonic ectoderm was also assessed. Explants were cultured in RPMI
+ 1 /*g/ml colcemid for 2 h at 37 °C, either directly after dissection from the
186
J. ROSSANT AND L. OFER
embryo or after 1 or 2 days in culture. The fragments were then fixed in acetic
alcohol (1 part acetic acid/3 parts ethanol) and dissociated on to glass slides
in 60 % acetic acid (Evans, Burtenshaw & Ford, 1972). The cells were mounted
and stained in a toluidine blue/mountant mixture (Breckon & Evans, 1969)
and the number of metaphases and interphase nuclei was counted. For smaller
explants, total cell counts were made but for larger fragments the slides were
scanned at intervals and all cells in each scan were counted.
Microdensitometry
Cell samples for microdensitometry were prepared by clearing away all
feeder cells from the edges of the explants and then trypsinizing the embryoderived cells from the plastic. The trypsinized cells were dried on to a clean
glass slide and fixed in acetic alcohol for 1-8 h. After washing in absolute
ethanol the slides were stored dry and dust-free. A mouse liver imprint was also
placed on each slide and treated in the same way. The cell spreads were stained
by the Feulgen technique for DNA (Pearse, 1972). Microdensitometry measurements were made using a Quantimet 720 system (Image Analysing Computers
LW, Cambridge Instruments Ltd) with a Balzer K4 green interference filter,
peak transmission between 550 and 560 nm. A densitometer module was used
to digitize the optical density in steps of 0-02 absorbance units and sum these
for each picture point. The numerical value given is Zd/32 = D and the
absolute total integrated density is 32 x 0-02 x D.
Control liver readings were made for each slide. In all samples every cell in a
single scan was measured, but the whole of the sample was not always scanned.
The results were expressed in the form of histograms of total absorbance,
measured in arbitrary units. These histograms were then calibrated in multiples
of the haploid DNA value (C) by comparison with and extrapolation from the
liver controls, whose cells contain 2C, 4C, and 8 C values of DNA.
RESULTS
Ectopic transfers
The results of histological examination of the testes transfers are summarized
in Table 1. The success rate of extra-embryonic ectoderm grafts of all ages was
very high. All grafts identified were haemorrhagic and contained cells indistinguishable morphologically from trophoblast giant cells (Fig. 1). The extent
of the haemorrhage and the size and number of giant cells varied but the grafts
were similar to those produced by ectoplacental cones. Fewer embryonic grafts
were identified and none produced obvious haemorrhage. Histologically, these
grafts consisted of solid masses of relatively undifferentiated embryonic ectoderm-like cells (Fig. 2).
Properties of mouse extra-embryonic ectoderm
187
50 fim
•»*
Fig. 1. Section of haemorrhage produced by 8^-day extra-embryonic ectoderm
after 1 week under the testis capsule. Arrows indicate definite giant cells.
Fig. 2. Section of graft produced by 7^-day embryonic ectoderm after 1 week under
the testis capsule. Most cells appear similar to embryonic ectoderm.
188
J. ROSSANT AND L. OFER
Table 1. Tes'tis transfers of tissues from mouse egg cylinders,
examined after 7 days
Type of tissue identified
A
(
Type of
tissue
transferred
Age of
embryo
(days)
Extra-embryonic
ectoderm
51
61
71
81
51
61
71
81
61
71
Embryonic
ectoderm±
mesoderm
Ectoplacental
cone
No.
transferred
No.
haemorrhagic
grafts
No.
histologically
identified
8
5
11
7
10
10
9
7
4
1
5
5
10
7
0
0
0
0
4
1
5
5
10
7
2
4
8
4
4
1
Many
trophoblast Undifferentiated
giant cells
diploid cells
5
5
10
7
—
4
1
—
2
4
8
4
—
Table 2. In vitro culture of tissues isolated from mouse egg cylinders,
examined after 7 days
Predominant cell types in explants
Type of tissue
explanted
Age of
embryo
(days)
Extra-embryonic
ectoderm
Embryonic
ectoderm ±
mesoderm
Ectoplacental cone
51
61
71
81
51
61
71
81
51
61
71
81
No.
explanted
29
32
17
12
18
42
17
12
8
30
5
2
No. alive Trophoblast
after 7 days giant cells
25
27
16
11
3
13
17
11
3
28
5
2
Epithelial- Differentiating
like cells muscle and nerve
25
27
16
11
3
10
4
3
13
11
3
28
5
2
In vitro culture
The results of culture of embryonic and extra-embryonic fragments are
summarized in Table 2 and confirm the results of ectopic transfers. Again the
success rate of extra-embryonic ectoderm development was high and the cells
resembled those produced by ectoplacental cones in both size and morphology
after 1 week in culture. Embryonic ectoderm fragments often produced differentiating tissues but no morphologically giant cells were observed.
After 1 week in culture, the cell number of the extra-embryonic fragments did
Properties of mouse extra-embryonic ectoderm
189
Table 3. Mitotic index of tissues isolated from mouse egg cylinders after
various times in culture
Mitotic index (no. metaphases/no. cells x 100)
± standard error, after 2 h colcemid treatment
Type of tissue
explanted
Age of embryo
(days)
Extra-embryonic
ectoderm
5*
Embryonic
ectoderm
7*
8*
0 day explants
1 day explants
2 day explants
151 ± 3 0
16-8 ±1-4
20-9+11
10-1 ±3-2
13-9 ±0-8
11 0 + 0-4
2-4 ±1-2
3-4±l-5
3-4±0-l
l-6±0-3
10-4 ±0-7
9-5 + 1-3
0-4 ±0-2
21 ±01
0-9±0-6
19-3 ±3-8
8-9 + 0-6
0
Note: The mitotic index in each case is the average of at least three samples. The cell
number in each sample ranged from 60 to 1700.
not seem to have increased greatly, while the embryonic fragments were often
very large. This suggested that little if any cell division had taken place following
explanation of the extra-embryonic ectoderm fragments, and so the mitotic
index of these explants was examined early in the culture period. The results
are presented in Table 3. The mitotic index of the extra-embryonic ectoderm
when isolated from the embryo is as high or higher than that of the embryonic
ectoderm, but after only 24 h in culture it has dropped dramatically. After 2
days in culture the number of metaphases observed in the extra-embryonic
ectoderm explants is extremely low and in the 5^-day explants, cell division has
ceased entirely. The mitotic index remains high in the embryonic regions.
Microdensitometry
Microdensitometry measurements confirm that cells with high DNA contents are produced in vitro by isolated extra-embryonic ectoderm from 5^- to
8^-day embryos. The embryonic ectoderm explants produced no giant cells and
most cells had DNA values around 2C (Fig. 3). The peak was actually slightly
below the 2C level but this may be an artifact since no correction was made
for the obvious condensed nature of these nuclei (Goldstein & Bedi, 1974). It
can also be seen that contamination with tetraploid feeder cells is minimal. By
contrast, there are very few extra-embryonic ectoderm cells which fall into the
diploid class after 1 week in culture, and some reach DNA values of 128 C
or more (Fig. 4). The peak DNA value seems to be 8 C for all stages analysed
but the percentage of cells falling into the 0-8 C range varies with age at explantation. For 5^-day extra-embryonic ectoderm, 37 % of the cells fall into
the 0-8 C range, while at 6^ days, this percentage rises to 57 %. At 1\ days, the
percentage is 60 % and at 8^- days, it is 87 %. The larger proportion of cells
with DNA values greater than 8 C from 5^-day extra-embryonic ectoderm may
13
EMB
39
190
J. ROSSANT AND L. OFER
(A)
51-day embryonic ectoderm
4C
2C
(B)
200 -
2C
61-day embryonic ectoderm
4C
8C
120r
180 100 -
160
=
o
o
80 -
140
2 60
120
5 - 40
100
80
1
Ul_
25
50
75
100
Total absorbance
Total number of cells = 593
(Q
71 clay embryonic ectoderm
2C
4C
60
40
8C
20
0
100
0
80
25
50
75
Total absorbance
Total number of cells = 803
100
60
f
40
81 day embryonic ectoderm
25
50
75
Total absorbance
Total number of cells = 358
100
4C
8(
40
1 30 -
2C
.
I
4C
1
60
Liver control
2( 3
(D)
70
50
40
30
n
oo
S 20 -
20
1 JO
10
0
25
50
75
Total absorbance
Total number of cells = 216
100
25
Total absorbance
Total number of cells = 536
50
Properties of mouse extra-embryonic ectoderm
191
be related to the fact noted previously, that they cease cell division in culture
earlier than do explants from older embryos. Fig. 4E confirms that extraembryonic ectoderm cells are diploid before culture.
DISCUSSION
Extra-embryonic ectoderm isolated from the mouse embryo as late as 8^- days,
when the chorion is well developed, can form cells with the morphological
characteristics of trophoblast giant cells, both in ectopic sites and in vitro.
Microdensitometry measurements have shown that cells containing more than
128 x the haploid DNA value can develop from extra-embryonic ectoderm in
culture. The similarity between the properties of extra-embryonic ectoderm and
ectoplacental cones at all stages up to 8^ days provides further support for the
postulated common origin of these tissues from the trophectoderm of the
blastocyst (Gardner & Papaioannou, 1975).
Retention of the capacity to form giant cells by the isolated extra-embryonic
ectoderm, which may never produce giant cells in the intact embryo, suggests
that the mural giant cells and polar diploid trophoblast cells, which can first
be distinguished at the late blastocyst stage, may not represent two distinct
determined populations (Gardner & Rossant, 1976). There is no evidence that
giant trophoblast cells can ever return to the diploid, mitotically active state
(Zybina & Tikhomirova, 1963; Zybina & Mos'yan, 1967) but it is possible that
the diploid trophoblast cells always retain the capacity to produce giant cells.
It has previously been shown by many workers, notably Grobstein (1950), that
ectoplacental cones isolated as late as 12 days of gestation will produce
haemorrhage and giant cells when transferred to ectopic sites. However, such
isolated cones contain many secondary giant cells as well as diploid trophoblast
and it has not been proved conclusively that the diploid trophoblast cells can
transform into giant cells. The present results provide an example of known
diploid cells (Fig. 4E), of apparent trophectoderm origin (Gardner & Papaioannou, 1975), retaining the capacity to form giant cells as late as 4 days after
implantation. Later than 8^ days of gestation it becomes increasingly difficult
to separate the diploid trophoblast from the overlying giant cells in the placenta,
so that it is not yet known whether this property is retained by the diploid cells
beyond this stage.
It is not clear what maintains the extra-embryonic ectoderm in a diploid
Fig. 3. Histograms of DNA values of cells from explants of 5^ to 8^-day embryonic
ectoderm after 1 week in culture. Figure 3E shows a control liver sample, with cells
containing 2C, 4C and 8 C amounts of DNA. The labeled arrows on the other histograms mark the expected peak absorbance for a given C value, calculated from the
liver controls. All slides were treated together, and the liver controls were similar
in all; direct comparison between values of absorbance from different slides could
therefore be made. All histograms represent the pooled data from two samples.
13-2
192
J. ROSSANT AND L. OFER
(A) 51-day extra-embryonic ectoderm
oo o
cells
20 p
15 -
o
o
10 -
"i
5
3
(B) 65-day extra-embryonic ectoderm
oo o
o
\
HI,
o
I 1
\
100r
80
60
•
ol-
o
40
Ulfl|Ul«,Mnnnmn 1 In
1
0
1
500
1000
1500
Total absorbance
Total number of cells =127
n
2000
20
500
1000
1500
Total absorbance
Total number of cells = 583
(C) 74-day extra-embryonic ectoderm
(D) 8]- day extra-embryonic ectoderm
OO
O
250
500
750
Total absorbance
Total number of cells = 577
1000
(E) 1\ day extra-embryonic ectoderm-non-cultured
OO
rj
20
375
Total absorbance
Total number of cells = 584
15
L
10
5
ln£L
25
50
75
Total absorbance
Total number of cells = 152
100
Fig. 4. Histograms of DNA values of cells from explants of 5£ to 8i-day extraembryonic ectoderm after 1 week in culture. Haploid DNA (C) values were calculated from the liver control readings (Fig. 3E). Fig. 4E shows the DNA values of
cells from 7-^-day extra-embryonic ectoderm before culture. All histograms represent
the pooled data from two samples.
2000
Properties of mouse extra-embryonic ectoderm
193
state in the intact embryo. At the blastocyst stage, contact with the ICM is
thought to be necessary to maintain proliferation of the overlying trophectoderm cells, while cells away from the ICM endoreduplicate their DNA and
transform into giant cells (Gardner, 1971, 1972; Barlow & Sherman, 1972;
Gardner et ah 1973; Ansell & Snow, 1975). The fact that extra-embryonic
ectoderm ceases cell division rapidly when separated from the rest of the
embryo (Table 3) supports the suggestion that continued contact with ICM
derivatives is necessary to promote postimplantation trophoblast proliferation
(Gardner, 1975). This may be a specific inductive effect or the embryonic tissues
may simply serve to maintain the organization and close cell contacts of the
proliferating extra-embryonic ectoderm, since formation of giant cells by isolated extra-embryonic ectoderm is associated with disorganization of its normal
structure as well as isolation from embryonic tissues.
We should like to thank Drs R. L. Gardner and C. F. Graham for useful discussion and
Dr M. J. Evans and University College London for use of the microdensitometer. The
work was supported by the Medical Research Council. J.R. is a Beit Memorial Junior
Research Fellow.
REFERENCES
J. D. & SNOW, M. H. L. (1975). The development of trophoblast in vitro from
blastocysts containing varying amounts of inner cell mass. / . Embryol. exp. Morph. 33,
177-185.
BARLOW, P. & SHERMAN, M. I. (1972). The biochemistry of differentiation of mouse trophoblast: studies on polyploidy. /. Embryol. exp. Morph. 27, 447-465.
BRECKON, G. & EVANS, E. P. (1969). A combined toluidine blue stain and mounting medium.
In Comparative Mammalian Cytogenetics (ed. K. Benirschke), pp. 465-466. New York:
Springer-Verlag.
DIWAN, S. & STEVENS, L. C. (1975). Development of teratomas from the ectoderm of mouse
egg cylinders. Proc. Am. Ass. Cancer Res. 16, 98.
DUVAL, M. (1891). Le placenta des rongeurs. /. Anat. Physiol, Paris 27, 279-476.
EVANS, E. P., BURTENSHAW, M. D. & FORD, C. E. (1972). Chromosomes of mouse embryos
and new born young: preparations from membranes and tail tips. Stain Techn. 47, 229-234.
GARDNER, R. L. (197.1). Manipulations on the blastocyst. In Advances in the Biosciences,
No. 6, Schering Symposium on Intrinsic and Extrinsic factors in Early Mammalian Development (ed. G. Raspe), pp. 279-296. Oxford: Pergamon Press.
GARDNER, 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. (1975). Origins and properties of trophoblast. In The Immunobiology of
Trophoblast (ed. R. G. Edwards, C. W. S. Howe & M. H. Johnson), pp. 43-65. London:
Cambridge University Press.
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 Fdn. Symp.), 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. London:
Cambridge University Press.
ANSELL,
194
J. ROSSANT AND L. OFER
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. J. Embryol. exp. Morph. 30, 561-572.
GARDNER, R. L. & ROSSANT, J. (1976). Determination during embryogenesis. In Embryogenesis in Mammals (Ciba Fdn. Symp.), pp. 5-25. Amsterdam: Associated Scientific Publishers.
GOLDSTEIN, D. J. & BEDI, K. S. (1974). Cytophotometric factors causing apparent differences
between Feulgen DNA contents of different leucocytes. Nature, Lond. 251, 439-440.
GROBSTEIN, C. (1950). Production of intra-ocular haemorrhage by mouse trophoblast. /. exp.
Zool. 114, 359-373.
JENKINSON, J. W. (1900). A reinvestigation of the early stages of the development of the
mouse. Q. Jl Microsc. Sc. 43, 61-82.
JENKINSON, J. W. (1902). Observations on the histology and physiology of the placenta of
the mouse. Tijdschr. ned. dierk. Vereen. 7, 124-198.
LEVAK-SVAJGER, B., SVAJGER, A. & SKREB, N. (1969). Separation of germ layers in presomite
rat embryos. Experientia 25, 1311-1312.
PEARSE, A. G. E. (1972). Histochemistry. Theoretical and Applied, Volume 2. London:
Churchill and Livingstone.
ROBINSON, A. (1904). Lectures on the early stages in the development of mammalian ova and
on the formation of the placenta in different groups of mammals. I. /. Anat. Physiol. 38,
1-19.
SNELL, G. D. & STEVENS, L. C. (1966). Early embryology. In Biology of the Laboratory
Mouse (ed. E. L. Green), pp. 205-245. New York: McGraw-Hill.
WHITTINGHAM, D. G. & WALES, R. G. (1969). Storage of two-cell mouse embryos in vitro.
Aust. J. biol. Sci. 22, 1065-1068.
ZYBINA, E. V. & MOS'YAN, I. A. (1967). Sex chromatin bodies during endomitotic polyploidization of trophoblast cells. Tsitologiya 9, 265-272. (In Russian.)
ZYBINA, E. V. & TIKHOMIROVA, M. M. (1963). The question of endomitotic polyploidization
in giant cells of the trophoblast. In Cell Morphology and Cytochemistry, pp. 53-63. Moscow : Nauka. (In Russian.)
GARDNER,
{Received 21 October 1976, revised 3 February 1977)