/. Embryol. exp. Morph. Vol. 35, J,pp. 81-86, 1976
81
Printed in Great Britain
The immediate postimplantation development
of tetraploid mouse blastocysts
By M. H. L. SNOW1
From the MRC Mammalian Development Unit, University College London
SUMMARY
The development during and immediately after the implantation period of 143 tetraploid
blastocysts was studied both in vitro and in vivo; 58-7 % in vitro and 38-8 % in vivo were
found to exhibit the changes associated with the early implanting blastocyst, i.e. giant cell
transformation of the trophoblast and induction of the decidual cell reaction in the uterus.
Of these 38-7 % in vitro and 19-4 % in vivo showed evidence of inner cell mass function
during this time but only two in each system could be claimed as showing normal development. Examination of the developing blastocyst leads to the conclusion that lack of cell
numbers in the inner cell mass is the most likely reason for the poor development of tetraploid
embryos and suggests that the minimum number of ICM cells required to fulfil its role in
embryogenesis is between four and eight.
INTRODUCTION
The randomly bred Q strain of mice will tolerate tetraploidy to a far greater
extent than other mammals in which it has been recorded (Snow, 1973, 1975a).
Even so, only 6 % of tetraploid blastocysts, i.e. 19 % of those that implant, will
develop advanced embryos (Snow, 1975a). The immediate postimplantation
period, from A\ to 6^ days of development, has been examined in order to
determine what constraints may operate to prevent embryo formation in most
blastocysts. The behaviour of tetraploid blastocysts grown in vitro beyond the
implantation stage has also been studied, using the technique described by
Ansell and Snow (1975) in which the ability of the inner cell mass to control
trophoblast development is assessed.
MATERIALS AND METHODS
The procedures for creating tetraploid embryos have already been described
(Snow, 1973, 1975o). In vitro development of blastocysts was studied by the
technique of Ansell & Snow (1975), using Whitten's medium (Whitten, 1971)
supplemented with 5 % foetal calf serum. The substrata were microscope slide
cover-glasses cleaned by washing in N-HC1, 2 x distilled water and absolute
1
Author's address: MRC Mammalian Development Unit, Wolfson House, University
College London, 4 Stephenson Way, London NW1 2HE.
6
EMB
35
82
M. H. L. SNOW
ethanol and air-dried. In utero, A\- and 5-^-day implantation sites were identified
by the Pontamine sky blue reaction (Orsini & McLaren, 1967); later ones were
clearly visible as decidual swellings. All sites were excised and processed for
wax embedding. Sections, 6 or 10 pum, were stained with Cole's iodine-ripened
haematoxylin (Item 29, Cole 1943) and 0-25 % aqueous congo red.
Table 1. Summary of the cell count data on developing 4N blastocysts
in vitro and in vivo
(Different superscript letters following the total cell no. figures indicate a significant
difference between those figures (P < 005).)
No. embryos
scored
2N blastocysts
2N outgrowths
4N blastocysts
4N outgrowths, total
4N outgrowths with ICM
4N outgrowths with trophoblast
only
4N trophoblast vesicles in utero
•5
Total cell no.
(mean ± S.E.)
Trophoblast cell no.
(mean ± S.E.)
45
20
109
31
12
19
54-0±2-17a
79-8±3-2b
20-4±0-69c
220 ±20°
30-2±3-3fl
16-8±l-5e
20-6 ±1-7
26-5 ± 3 0
16-8 ±1-5
13
18-2±3-0c
18-2±30
—
67-5 ±3-8
—
1 0 -
o
2N
O
•C
5 ~
e
0
50
Number of cells
Fig. 1. A histogram showing the distribution of cell numbers in outgrowth from 4N
and 2N blastocysts.
RESULTS
In vitro. There was no significant variation between replicate experiments
either with respect to blastocyst production or to outgrowth performance.
Data have therefore been pooled and are shown in Table 1 and in Fig. 1.
All blastocysts that hatched also attached and outgrew. It was found that
29/30 (96-7 %) diploid and 37/63 (58-7 %) tetraploids produced outgrowths. For
Tetraploid egg cylinders
83
cell counting nine diploids were discarded: one (with 27 cells) was abnormal
with several pycnotic nuclei, six were damaged during processing and two
possessed such a large inner cell mass (ICM) that cell counting was impossible.
Similarly two abnormal (three and nine cells) and four damaged tetraploids
were discarded. In both diploid and tetraploid outgrowths trophoblast cells
can be recognized on morphological criteria (Ansell & Snow, 1975). All diploid
outgrowths possessed ICM components but in 19 of the tetraploids ICM cells
were absent.
Table 2. The results of transfer of 4N blastocysts to
pseudo-pregnant foster mothers
Age
(days)
4i
H
6k
Totals
No. recipients No. pregnant
No. blastocysts
transferred
No. implantations No. embryos*
1
1
6
2
2
14
7
6
60
9
80
10
* Includes all implants showing evidence of ICM
6
4
21
31
development.
1 (?)
0
5
6
In utero. The data are shown in Table 2 and Fig. 2. Not all embryos were
normal. The 4^-day 'embryo' consisted of about six 'inside' cells within the
blastocyst cavity; they were not grouped together as an ICM. Two of the
6^-day embryos appeared normal (Fig. 2 A, B) although of different sizes. Of
the others, one showed reduced and disorganized embryonic ectoderm (Fig.
2C), another had its embryonic component rounded up as a sphere and
detached from a disaggregated extra-embryonic region (Fig. 2D) and the third
was extremely small although all components could be identified (Fig. 2E).
In 21 of the remaining 24 decidua only trophoblast cells were found. In three
(all 6\ days) only cell debris and blood could be seen. In 13 of the decidua
containing only trophoblast, cell counts were possible from serial sections (see
Table 1). In the 4^-day decidua containing only trophoblast, giant cell transformation had already occurred in many trophoblast cells. This represents very
precocious development.
DISCUSSION
These data show that many tetraploid blastocysts lack a functional inner cell
mass. The high proportion of trophoblast-only blastocysts in early implantation
sites (67 %) suggests that they and the trophoblast-only outgrowths (58 %) are
derived from blastocysts devoid of inner cell mass altogether. However, the
outgrowth medium is not designed to facilitate ICM growth and it is probable
that some blastocysts actually lose their ICM during the culture period. Similarly, in utero by 6^ days some ICMs may have perished, although no evidence
6-2
84
M. H. L. SNOW
100 fim
H
Endoderm
[13 Embryonic ectoderm
ED Extra embryonic ectoderm
Fig. 2. Outline camera lucida drawings of midline sections of the five 6^-day 4N
'embryos'. Symbols drawn as having a common origin according to the cell lineage
studies of Gardner & Papaioannou (1975). r. = Reichert's membrane; intact in all
embryos but only drawn complete in D.
Tetraploid egg cylinders
85
for dead ICMs was found. Nevertheless, it seems likely that many tretraploid
blastocysts should more accurately be described as trophoblastic vesicles, and
could not yield embryos. Further, it seems from the numerical data that not all
tetraploids with ICM components are capable of sustaining embryonic development in utero. Perhaps there is a critical minimum number of ICM cells necessary to control trophoblast activity such that the normal synchrony between
conceptus development and uterine changes is maintained, and successful
embryogenesis is achieved. Gardner (1972) has suggested that the ICM is
required for trophoblast proliferations in vivo. Ansell & Snow (1975) and Snow
(19756) extended these observations to indicate that in vitro the ICM apparently
controlled the rate at which trophoblast growth and differentiation occurred.
How many ICM cells are required to make an embryo? Gardner (1971) has
demonstrated that single inner cell mass cells injected into mouse blastocysts
can result in a high degree of chimaerism at later stages, suggesting that the
number of ICM cells involved in embryo formation can be small. If it is
assumed, on the evidence of the in vivo data, that 6 % of tetraploid blastocysts
can develop into advanced embryos this would correspond in the present study
to two of the 31 tetraploid blastocysts used for outgrowth studies. The two
tetraploid outgrowths that showed the greatest trophoblast proliferation were
in fact significantly different from the remaining tetraploid outgrowths by a
statistical outlier determination (P < 0-05). The remaining 29 outgrowths seem
to form a single population. The two outliers had ICMs composed of eight and
five cells. The average tetraploid blastocyst would be expected to contain 5-7
ICM cells on the assumption that the ICM constitutes 28 % of the cells in a
blastocyst (Barlow, Owen & Graham, 1972; Horner & McLaren, 1974).
The hypothesis explaining ICM/trophoblast differentiation currently in
favour states that those cells which during cleavage lose contact with the external
environment and become totally enclosed by other cells are determined to form
ICM. Cells which remain 'outside' will form trophoblast (Graham, 1973;
Gardner, 1974; Herbert & Graham, 1974). The simple model of packing
spheres of equal size shows that at least 17 are needed to have one completely
enclosed (Izquierdo & Ortiz, 1975). The progeny of such an inside cell at the
blastocyst stage would number between four and eight cells. The tetraploid
data support the idea that four to eight cells is the lower limit for a functional
ICM.
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M. H. L. SNOW
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{Received 3 July 1975; revised 15 October 1975)