/ . Embryol. exp. Morph. Vol. 35, J, pp. 95-106, 1976
95
Printed in Great Britain
The effect of short-term labelling in
[ H]thymidine on the viability of mouse
blastomeres: alone and in combination
with unlabelled blastomeres
3
By SUSAN J. KELLY 1 AND JANET ROSSANT 1
From the Department of Zoology, Oxford
SUMMARY
A method of labelling 8-cell-stage mouse blastomeres with [3H]thymidine is described,
which allows them to be followed to the late blastocyst stage and is compatible with normal
postimplantation development. However, the [3H]thymidine does affect the postimplantation
vigour of the cells when placed in competition with unlabelled cells in blastomere aggregates.
This suggests that caution should be used in interpreting results using [3H]thymidine as a
label for early mammalian cells.
INTRODUCTION
In mammalian embryology, tritiated thymidine has been widely used as a
marker for studying DNA synthesis (e.g. Mintz, 1964a, 1965; Samoshkina,
1968; Piko, 1970; Barlow, Owen & Graham, 1972) and for following the fate
of cells in blastomere aggregation experiments (Mintz, 19646; Hillman, Sherman
& Graham, 1972; Garner & McLaren, 1974). However, culture of 2-cell mouse
embryos for long periods (12-70 h) in low concentrations of [3H]thymidine
(0-01-0-1 /tCi/ml) has been shown to have a deleterious effect on their development (Snow, 1973a, b). Snow found a sharp reduction in cell number at the 16to 32-cell stage, and embryos that developed into blastocysts showed little or no
inner cell mass (ICM) formation. These trophectoderm vesicles could implant
when transferred to pseudopregnant recipients, but the resulting decidua contained only a few trophoblast giant cells and no ICM derivatives. Thus, the
implicit assumption of previous workers, that [3H]thymidine did not interfere
with development in the experiments they performed, may not be valid. Most
authors did not compare the development of labelled and unlabelled blastomeres
in their experimental systems, with the exception of Garner & McLaren (1974).
They found that cell number in early blastocysts was not reduced by incubation
in 0-01 /tCi/ml [3H]thymidine from the 2-cell to the 8-cell stage.
1
Authors' address: Department of Zoology, South Parks Road, Oxford, U.K. (Reprint
requests to Janet Rossant.)
96
S. J. KELLY AND J. ROSSANT
In a study specifically designed to test the viability of blastomeres after [3H]thymidine treatment, Horner & McLaren (1974) reported that embryos cultured
continuously from the 2-cell stage in concentrations as low as 5 nCi/ml failed
to develop normally after implantation, although cell number was not reduced
at the blastocyst stage. Similar culture in 1 nCi/ml allowed normal postimplantation development in 40-5 % of cases, but the level of labelling achieved by this
procedure was not high enough to be useful for following cell behaviour in
developing blastocysts (mean grain count/nucleus = 1-6, after three days in
culture). In this respect even the labelling procedure of Garner & McLaren
(1974) has limited application since they attained levels of labelling which could
only be followed through two cell divisions to the 32-cell stage, but were too
weak for labelled cells to be detected at the 64-cell stage. It is therefore important
to determine whether a labelling procedure may be devised which allows blastomeres to be labelled sufficiently highly at the 8-cell stage to permit their fate to
be followed to the mature blastocyst stage and which is also compatible with
normal postimplantation development.
In various recent studies, we have used a procedure by which blastomeres
may be labelled during the S-phase of the 8-cell stage at a level which allows
their division products to be identified readily three or even four cell divisions
later, at the late blastocyst stage. The viability of such labelled blastomeres was
tested in two ways. Firstly, blastocysts derived from totally labelled 8-cell-stage
embryos were transferred to pseudopregnant recipients to see if they would
develop into normal postimplantation embryos. Secondly, labelled blastomeres
were combined with unlabelled blastomeres of a different genotype for the
isozymal variants of glucose phosphate isomerase (GPI-1) and the resulting
chimaeric blastocysts transferred to pseudopregnant recipients. The contribution
of the labelled blastomeres to any resulting conceptuses could be assessed by
the presence of the appropriate GPI-1 isozyme. This competitive situation
provides a very stringent test of their viability and vigour.
MATERIALS AND METHODS
(1) Source of embryos
All embryos were obtained from spontaneously ovulating mice. Embryos
either homozygous aa or heterozygous ab at the Gpi-1 locus (De Lorenzo &
Ruddle, 1969) were obtained from females of the PO stock (a random-breeding
Swiss albino mouse from the Pathology Department, Oxford) mated with
CFLP males (Anglia Laboratory Animals Ltd), known to be homozygous aa
for Gpi-1. Embryos homozygous for the b allele of Gpi-1 were obtained from
mice of the inbred strain C57BL/6J. The day that a copulation plug was found
was designated day 1 of pregnancy.
Viability of labelled mouse blastomeres
97
(2) Dissociation and labelling of embryos
Embryos were recovered from the oviduct at the 4- to 8-cell stage on the
morning of day 3. All embryos were recovered, stored and cultured in Whitten's
medium (Whitten, 1971), equilibrated with a 5% O2, 5% CO2, 90% N 2 gas
mixture and kept at 37 °C. Zonae were removed by pronase digestion (in dialysed,
prewarmed, 0-25% pronase - Mintz, 1962) and the embryos were allowed to
recover in Whitten's medium for a short period.
PO embryos at the 4-cell stage were dissociated into their component blastomeres by gently sucking in and out of a flame polished micropipette. Each
blastomere was then placed in a separate drop of culture medium under paraffin
oil and observed every 30 min. When the blastomeres had divided to the 8-cell
stage, some were left in culture and some were placed in Whitten's medium
supplemented with [3H]thymidine (specific activity 26 Ci/mM, Radiochemical
Centre, Amersham, U.K.) at 0-25 /tCi/ml for 2 h. At the end of the 2-hour
period the blastomeres were removed and placed in microdrops of Whitten's
medium supplemented with 50% foetal calf serum. They were removed at
intervals during the first hour to several successive drops and then cultured for
3-5 h to dilute the unincorporated label. At the end of this period they were
rinsed in Whitten's medium and returned to microdrops of this medium.
(3) Reaggregation of blastomeres
Four types of reaggregates using pairs of 8-cell-stage PO blastomeres were
made.
(1) Four pairs of labelled PO blastomeres, forming a totally labelled embryo.
(2) Four pairs of unlabelled PO blastomeres, forming an unlabelled embryo.
(3) One pair of labelled PO blastomeres with six 8-cell stage C57BL/6 blastomeres {unlabelled) (see Fig. 1).
(4) One pair of unlabelled PO blastomeres with six 8-cell stage C57BL/6
blastomeres {unlabelled) (see Fig. 1).
All aggregates were cultured to the blastocyst stage.
(4) Autoradiography
Fully labelled and composite blastocysts were fixed for cell counts and autoradiography. Following a rinse in phosphate-buffered saline, blastocysts were
fixed in fresh Heidenhain's fixative (Tarkowski & Wroblewska, 1967). They
were embedded in agar (Hillman et al. 1972) which was then treated as a large
specimen for embedding in wax. Serial sections were cut at 5 ftm and, after
location of the blastocysts, the slides were dipped in llford K2 emulsion and
exposed for 2 weeks. After developing, the slides were stained with haemalum
and light green. The number of cells in each fully labelled blastocyst and the
numbers of labelled and unlabelled cells in each composite blastocyst were
counted.
7
EMB
35
98
S. J. KELLY AND J. ROSSANT
(5) Transfers of the blastocysts
Groups of fully labelled PO blastocysts were transferred to one uterine horn
of a pseudopregnant foster mother and, where possible, unlabelled PO blastocysts were transferred, as controls, to the other. Similarly, groups of composites
made with labelled PO blastomeres were transferred, using composites made
with unlabelled PO blastomeres as controls. Transfers were performed on the
evening of the third or the morning of the fourth day of pseudopregnancy.
In addition, one or two experimental blastocysts from each transfer experiment were treated for autoradiography as described above to check the efficacy
of the labelling regime.
Fig. 1. Arrangement of blastomere composites at the 8-cell stage. Two PO blastomeres are surrounded by six C57BL/6 blastomeres.
(6) Assessment ofpostimplantation development of fully labelled embryos
Fully labelled embryos and unlabelled controls were treated in two ways.
One group was recovered on day 6 and the implants fixed in AFA, mounted in
wax and sectioned for histological examination. The other group was recovered
on day 10 and the implants dissected and scored for the presence of embryonic
derivatives.
99
Viability of labelled mouse blastomeres
(7) Gpi-1 analysis of transferred composites
Composite embryos recovered on day 10 were separated into three fractions:
embryo, embryonic membranes and trophoblast. Each fraction was analysed
for the isozymes of GPI-1 (Chapman, Whitten & Ruddle, 1971). One series of
composites using unlabelled PO blastomeres was recovered at term.
I II I
50 fm
Fig. 2. Section of fully labelled PO blastocyst. (Two cells of an adjacent blastocyst
appear to the right.)
RESULTS
(1) Autoradiography and cell counts at the blastocyst stage
Blastomeres fixed at this stage have gone through up to three divisions subsequent to labelling at S-phase of the 8-cell stage. Nevertheless, the levels of
labelling are good (often more than 30 grains per nucleus) and labelled cells are
readily identified (see Figs. 2 and 3).
Cell counts were made for the fully labelled blastocysts and unlabelled control
blastocysts. The mean number for labelled blastocysts is 47-2 (range 32-59),
7-2
100
S. J. KELLY AND J. ROSSANT
B
II MM
I
50 fan
'
Fig. 3. Sections of blastocysts derived from composites made of two labelled PO
blastomeres and six unlabelled C57BL/6 blastomeres. Arrows indicate labelled
cells. A and B are adjacent sections of the same blastocyst.
which is similar to the means found in unlabelled blastocysts cultured for similar
periods, 47-6 (range 36-59) for PO embryos and 41-9 (range 32-58) for C57BL/6
embryos.
Counts of labelled and unlabelled cells in the composites are shown in Table 1.
In 16 out of 17 blastocysts, the labelled cells made up less than one quarter of
the total number of cells, but the range was wide. The labelled cells were found
in both ICM and trophectoderm of most blastocysts, and were morphologically
indistinguishable from the unlabelled cells (see Fig. 3).
101
Viability of labelled mouse blastomeres
Table 1. Numbers of labelled and unlabelled cells in composite embryos
fixed at the blastocyst stage
No. labelled
A
Blastocyst
no.
1
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
Range
Average
Total cell
no.
1CM
47
40
54
51
55
57
52
60
63
59
47
49
50
58
44
52
64
40-64
53
Trophectoderm
3
3
2
2
3
2
4
4
2
4
2
0
4
3
1
2
2
—
10
13
10
9
10
4
10
7
8
13
—
—
—
5
5
8
11
9
9
7
Total
No.
unlabelled
No. labelled/
total cell no.
8
8
10
13
12
11
11
14
15
14
11
10
8
13
8
10
15
8-15
112
39
32
44
38
43
46
41
46
48
45
36
39
42
45
36
42
49
32-49
41-8
0-17
0-20
018
0-25
0-22
019
0-21
0-23
0-24
0-24
0-23
0-20
016
0-22
018
019
0-23
016-0-25
0 21
Table 2. Postimplantation development ofPO embryos labelled with [sH]thymidine
at S-phase of the 8-cell compared with the development of unlabelled controls
(only pregnant females considered)
[3H]thymidine
No. recovered/
no. transferred
at day 6
No. recovered/
no. transferred
at day 10
0-25 /iCi/ml
3/6
3/6
2/2
4/4
(2) Postimplantation development of fully labelled embryos
Normal embryonic development was found at day 6 and day 10 following
transfer of fully labelled PO blastocysts and unlabelled controls (Table 2, Fig. 4).
(3) GPI-1 analysis of composites recovered at day 10
The GPI-1 analysis of 13 composites recovered on day 10, which had been
made with labelled PO blastomeres, is shown in Table 3. Only one embryo (3)
shows a PO-derived contribution to the embryo and membranes fraction. A
further four (1, 10, 11, 13) show contributions to the trophoblast fractions.
A comparable analysis of composites made with unlabelled PO blastomeres is
A'.' * C
B
Fig. 4. A, Section of day-6 implant derived from a fully labelled PO blastocyst.
B, Section of day-6 implant derived from an unlabelled PO blastocyst.
00
o
t- 1
t-
w1
C/3
Viability of labelled mouse blastomeres
103
Table 3. Embryos recovered at day 10 from composites made at the 8-cell stage
with two [3H]thymidine-/abelled PO blastomeres and six unlabelled C57BLJ6
blastomeres
GPI-1 analysis
Embryo no.
1
2
3
4
5
6
7
8
9
10
II
12
13
Embryo
bb
bb
Membranes
Trophoblast
bb + ab
bb
bb
bb + ab
bb + (ab)
bb*
—
bb
bb*
bb*
bb*
bb*
bb*
bb
bb*
—
bb
bb*
bb*
bb*
bb*
bb*
bb
bb
bb
bb
bb
bb
bb
bb
bb
aa
bb + ab
bb
bb + aa
— Data not available.
* Embryo and membranes analysed together.
( ) Contribution less than 10 % of total activity.
Table 4. Embryos recovered from composites made at the 8-cell stage with two
unlabelled PO blastomeres and six unlabelled C57BLJ6 blastomeres
GPI-1 analysis
Embryo no.
Embryo
Membranes
Trophoblast
1
aa
bb + aa
bb + aa
2
3
4
(bb) + aa*
bb+(aa)*
(bb)+aa
(bb) + aa*
bb + (aa)*
(bb)+aa
—
—
—
—
—
—
—
—
—
—
—
bb + aa
(bb) + aa
(bb) + aa
—
—
—
—
—
—
—
—
—
—
—
5t
6t
7t
8t
9t
lot
lit
12t
13t
14t
15t
aa
aa
aa
(bb) + aa
aa
(bb) + aa
aa
aa
aa
(bb)+aa
aa
— Data not available.
* Embryo and membranes analysed together.
( ) Contribution less than 10 % of total activity.
t Embryo recovered at term. PO contribution assessed by GPI-1 analysis and by eye
and coat colour.
104
S. J. KELLY AND J. ROSSANT
shown in Table 4. All embryos showed PO-derived contributions; at day 10,
when membranes and trophoblast fractions were available, these also showed
PO-derived contributions.
DISCUSSION
A method of short-term incubation in [3H]thymidine has been devised which
produces a level of label sufficient to follow cells through three or four cell
divisions (Figs. 2 and 3). This method does not apparently reduce blastocyst
cell number and, more importantly, does not prevent the labelled cells from
forming live postimplantation embryos. A total of 7/10 live embryos was
obtained from blastocysts which had been fully labelled by this method. In
this small series no comment could be made on the relative implantation rate of
the labelled and unlabelled control embryos. Nevertheless, these results establish
that [3H]thymidine-labelled blastomeres are capable of normal postimplantation
development.
When two labelled FO blastomeres were combined with six unlabelled C51BL/6
blastomeres there was again no marked effect on cell number at the blastocyst
stage. Labelled cells generally contributed just less than one quarter of the total
cell number (Table 1). This represents a slight deviation from the expected
proportion (one quarter) of the total cell number, which may or may not be a
real effect, since labelled and unlabelled cells were derived from different strains.
In these composites labelled cells were usually found in both the ICM and the
trophectoderm (Fig. 3, Table 1).
When such composites were recovered at day 10 only 1 out of 11 embryo
fractions and 4 out of 13 trophoblast fractions analysed contained PO contributions (Table 3). This contrasts strongly with the results from the composites
of two unlabelled PO blastomeres and six unlabelled C57BL/6 blastomeres,
where all conceptuses showed PO contributions to the embryo fractions (Table
4). Also all four conceptuses analysed at day 10 showed PO contributions to
the trophoblast fractions. These results suggest that [3H]thymidine has a deleterious effect on the labelled PO blastomeres which only becomes apparent when
the blastomeres are placed in a competitive situation.
The /^-irradiation emitted by [3H]thymidine incorporated into DNA is known
to produce a variety of deleterious effects on cells (see review by Wimber, 1964).
Two of these effects are chromosome breakage and slowing of the mitotic rate.
Chromosomes breakage, if not extensive enough to cause cell death, may be
repaired. Similar damage caused by X-irradiation is known to be repaired in
bacterial (Town, Smith & Kaplan, 1974) and mammalian cells (Wolff, 1972).
Repeated repair of damage and rapid dilution of the label during development
may explain why normal postimplantation embryos have been obtained from
labelled cells in our present experiments. In studies on chick embryos, it has been
shown that normal embryonic development is not impaired after [3H]thymidine
labelling at various stages (Sauer & Walker, 1961; Weston, 1963).
Viability of labelled mouse bias tome res
105
3
Slowing of mitotic rate caused by [ H]thymidine provides a possible explanation for the poor development of labelled PO blastomeres in competition with
unlabelled cells. While blastocyst cell number is not markedly reduced in fully
labelled embryos, a slight retardation in mitotic rate may be indicated by the
significant deviation from the expected proportion of labelled to unlabelled cells
in composites (Table 1). At implantation a rapid increase in cell number occurs
(Buehr & McLaren, 1974), and if the labelled cells are in fact slightly retarded,
they will be strongly selected against during this mitotic burst. This will result
in the labelled cells being more or less eliminated from the later conceptus. Presumably, when the labelled cells are in a non-competitive situation,
such a slight delay prior to implantation is not sufficient to prevent continued
development.
These results suggest that caution should be used in interpreting experiments
where [3H]thymidine-labelled cells are placed in competition with unlabelled
cells. Such a competitive effect might be reduced by lowering the concentration
of the [3H]thymidine by half or reducing the time in labelled medium, either of
which should be possible in view of the high level of labelling achieved by the
present method.
We should like to thank Drs H. Alexandre, R. L. Gardner and C. F. Graham for valuable
discussion during the preparation of this manuscript. Both authors were in receipt of Medical
Research Council Research Studentships.
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