J. Embryol exp. Morph. Vol. 66, pp. 91-108, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Tetraploidy and early
development: effects on developmental timing and
embryonic metabolism
By MARTIN A. EGLITIS 1 AND LYNN M. WILEY2
From the Department of Anatomy, University of Virginia
SUMMARY
The effect of balanced gene dosage changes on the timing of cavitation and on the timing
of appearance of a stage-specific embryonic cell surface antigen was studied in preimplantation
mouse embryos. Gene dosage was increased by creating tetraploid embryos at the 4-cell stage,
either by blastomere fusion with polyethylene glycol (PEG) or by incubation in cytochalasin B
(cytB) to block cell division. Removal of the zona pellucida with Pronase from diploid
embryos caused a 7 h delay in cavitation. Further manipulations, either with PEG or cytB to
induce tetraploidy, did not produce a statistically significant additional delay in cavitation
timing. Likewise, PEG-induced tetraploidy did not affect the timing of appearance or disappearance of the embryonic cell surface antigen as compared with diploid control embryos.
In analysing the metabolic effects of tetraploidy, we found that in tetraploid embryos with cell
number equivalent to intact diploid embryos, MDH activity did not double with the doubling
of the genome, being only 50 % greater than diploid levels in cytB-induced tetraploid embryos
and only 20 % greater than diploid levels in PEG-induced tetraploid embryos. However, in
tetraploid embryos with one-half normal cell number, enzyme activity was equal to that in
whole diploid embryos, suggesting that in such embryos, MDH activity increased in parallel
with increases in gene dosage. Further studies showed that levels of RNA synthesis in PEGinduced tetraploid embryos also did not increase in parallel with the doubling of the genome.
Rather, these results suggested that in tetraploid embryos, compensation was made for at
least part of the excess genetic material.
INTRODUCTION
Although preimplantation development in the mouse is defined by numerous
morphological, biochemical, and physiological changes, there still remains the
problem of how their timing and coordination are controlled. This study was
initiated to investigate the control of the timing of formation of the blastocoele
(i.e. cavitation).
Recent hypotheses implicate the nucleus in the timing of preimplantation
development. The maternal uterine environment cannot be providing any
critical cues, since development in vitro proceeds normally and at very nearly the
1
Author's address: Department of Cell Biology, Roche Institute of Molecular Biology,
Nutley, New Jersey 07110, U.S.A.
2
Department of Human Anatomy, University of California, Davis School of Medicine,
Davis, California 95616, U.S.A.
92
M. A. EGLITIS AND L. M. WILEY
rate observed in vivo. Likewise, cytokinesis per se must not be critical in the
control of timing, since the number of cells and cell divisions can be altered
without affecting the timing of blastocyst formation (Smith & McLaren, 1977).
However, neither the number of nuclear divisions (i.e. DNA replications) nor
changes in the ratio of nuclear-to-cytoplasmic volume have been excluded from
consideration as possible repositories of the putative cue for cavitation (Smith &
McLaren, 1977; Alexandre, 1979; Braude, 1979; Surani, Barton & Burling,
1980).
The embryonic genome is known to be active during preimplantation development, as shown by the detection of mRNA synthesis (Knowland & Graham,
1972) and the expression of paternal isozymes (Chapman, Whitten & Ruddle,
1971; Wudl & Chapman, 1976). Also, the inhibitor of mRNA synthesis, aamanitin, has been used to show that blastocyst formation is dependent on new
mRNA synthesis (Braude, 1979). Since the genome is clearly functioning in
early development, it seems reasonable to study the nature of the 'developmental
clock' by determining whether nuclear changes will affect the timing of cavitation.
In this report, balanced increases in gene dosage to a tetraploid level were
used to determine whether they would alter the timing of cavitation. We produced tetraploid embryos with a recently developed method to fuse embryonic
blastomeres using polyethylene glycol (PEG; Eglitis, 1980), as well as by the
standard procedure using cytochalasin B (cytB; Snow, 1973). The interaction of
tetraploidy with cell number changes and its effect on developmental timing were
analysed by monitoring cavitation rates and the expression of a stage-dependent
embryonic cell surface antigen. Additional biochemical studies were done to
monitor the effect of tetraploidy on embryonic metabolism.
MATERIALS AND METHODS
Embryos at the 2-cell stage (47 ~ 49h after hCG injection) were flushed from
the oviducts of superovulated, random-bred DUB: (1CR) female mice with
bicarbonate-free modified Hank's balanced salts solution (BSS; Spindle &
Goldstein, 1975). Embryos were grown in modified (Spindle & Goldstein,
1975) standard egg culture medium (Biggers, Whitten & Whittingham 1971)
under paraffin oil (Fisher) and incubated at 37°C in a humidified atmosphere
of 5%C0 2 inair.
Depending upon the particular experiment, zonae pellucidae were removed
either from diploid 2-cell embryos before experimental manipulations (for
blastomere fusion) or from tetraploid 2- cell embryos after experimental manipulations (for cytB). Zonae pellucidae were removed by a 5 min incubation at
37 °C in 0-5 % protease (Pronase, Sigma; Mintz, 1962).
Tetraploid embryos were obtained by two methods: blastomere fusion
(Eglitis, 1980), or, inhibition of cell division with cytB (Snow, 1973). Briefly,
Development oftetraploid mouse embryos
93
(1) 48 hpost-hCG
(2) Pronase XjjJ^
8 h incubation \^M^
(4) Ca2+-free
disaggregation
(5)_PHA
(6) 45% PEG 1000V3iii7 (7) PHA
2 min
Fig. 1. A diagram illustrating the general features of the blastomere fusion protocol.
For a full description, see Eglitis (1980).
fused blastomeres were produced by disaggregating 4-cell-stage embryos,
aggregating individual blastomeres in pairs with phytohaemagglutinin (PHA,
Mintz, Gearhart & Guyment, 1973), and treating aggregated pairs for 2 min
with 45 % (w/v) PEG (Sigma, MW 1000; see Eglitis, 1980; Fig. 1). Alternatively,
tetraploid embryos were produced by incubating 2-cell embryos for 13 h (from
49 - 62 h post-hCG) in standard egg-culture medium containing 10 /*g cytB
(CalBiochem) per ml of medium.
To investigate the affect on developmental timing of the interaction of tetraploidy and altered cell number, both fusion and cytB-induced tetraploid
blastomeres were aggregated with PHA in various cell number combinations.
Fused blastomere tetraploid embryos (PEG-4N) were followed after aggregation
to restore cell number to that of equivalent (i.e. 4-cell) intact diploid embryos.
Alternatively, development of such tetraploid embryos with only half (\ PEG4N) or one-quarter (£ PEG-4N) the cell number of intact embryos was also
monitored. CytB-induced tetraploid embryos were followed either as 'halfembryos with ( | w/Z-cytB-4N) or without {\ ZF-cytB-4N) zonae pellucidae, or,
after zona removal, as aggregated to restore cell number to that of equivalent
intact diploid embryos (cytB-4N).
Controls consisted of intact diploid embryos with (w/Z-2N) or without
(ZF-2N) zonae pellucidae, as well as diploid embryos disaggregated at the 4-cell
stage to \ {\ ZF-2N) or \ (£ ZF-2N) diploid embryos. Additional controls
included blastomeres which did not fuse after PEG treatment, either reaggregated to restore cell number to that equivalent to intact embryos (PEG-2N)
or grown without reaggregation (\ PEG-2N).
Two parameters were used to time development of the embryos: (1) timing of
blastocoele formation, and (2) timing of expression of a stage-specific embryonic
cell surface antigen.
4
EMB 66
94
M. A. EGLITIS AND L. M. WILEY
To time progress towards cavitation, embryos were checked at intervals
through an inverted phase-contrast microscope. Embryos were scored as cavitating when an intraembryonic cavity became visible. The timing of cavitation was
compared between different classes of embryos by comparing their t$ of cavitation (i.e. the time at which 50 % of the final maximum proportion of cavitation
embryos was reached). To determine the t$, the linear regression was taken over
the times at which the proportion of cavitated embryos was uniformly increasing. Then, the t$ was calculated by finding the time at which one-half the final
maximum number of embryos had cavitated.
The timing of expression of the embryonic cell surface antigen was monitored
by indirect immunofluorescence (IIF). The particular antigen assayed was one
detected by an antiserum raised to 8-cell-stage embryos in a male New Zealand
white rabbit. The cell surface expression of this antigen (or antigens) is very
'stage-specific' in that it is restricted to the period immediately preceding and
following the onset of cavitation (around 94-106 h post-hCG). If necessary,
zonae pellucidae were mechanically removed immediately before the embryos
were observed through a Zeiss phase-contrast microscope equipped with
epifluorescence. Negative controls consisted of embryos incubated in the antiembryo antiserum at times when the antigen was not expressed, as well as
embryos incubated in pre-immune (normal) rabbit antiserum.
Two parameters were measured to assess the metabolic levels of tetraploid
embryos. The first was the determination of the level of activity of the constitutive enzyme, malate dehydrogenase (MDH). The level of MDH activity was
determined in diploid and tetraploid morulae (90 h post-hCG) and blastocysts
(120 h post-hCG) spectrophotometrically from the rate of reduction of NAD
(Brinster, 1966). Enzyme activity in moles NAD reduced/h/embryo was
calculated using the formula:
. . .
_
changes in absorbance/min x volume (0-1 ml) = 60 min
extinction coefficient (6-22) x 106 M x number of embryos.
In the absence of substrate, embryos reduced only an insignificant amount of
NAD.
The second measure of embryonic metabolism was the determination of the
level of RNA synthesis per blastomere. This was accomplished autoradiographically by quantitating incorporation of [3H]uridine into nuclear RNA.
Late morulae, (92-93 h post-hCG) were incubated for 3 h in standard egg
culture medium containing 0-5 or 0-05 /iCi/ml of [6-3H]uridine (specific activity
22-4Ci/mM, New England Nuclear; concentration 2-23 x 10"5 or 2-33 x 10~6
mM, respectively). Labelled embryos were rinsed thoroughly in BSS containing
2-33 x 10~2 mM cold uridine. After labelling, embryos were fixed onto glass slides
by a modification (Epstein, Smith, Travis & Tucker, 1978) of Tarkowski's
(1966) air-drying method. The slides were dried overnight in a dessicator at 4 °C
and then dipped in NTB-2 emulsion (Kodak); coated slides were allowed to dry
Development of tetraploid mouse embryos
95
Table 1. Timing of cavitation, t± = h post-hCG
Cell number class
Embryo type
Whole
Fused blastomere Tetraploids
Cytochalasin B Tetraploids
Diploid controls
w/Z-2N
96-9
r = 0-9766
PEG-4N
103-2
r = 0-9836
CytB-4N
105-9
r = 0-9877
ZF-2N
103-7
r = 0-9922
Half
Quarter
i PEG-4N i PEG-4N
100-9
99-8
r = 0-8952 r = 0-9374
\ CytB-4N
1020
r = 0-9979
i ZF-2N
i ZF-2N
104-9
104-2
r = 0-9633 r = 0-9795
r = Correlation coefficient calculated from Fig. 2.
in safe boxes. Embryos labelled in 0-5 ju,Ci [3H]uridine/ml were developed 18 h
after dipping, while embryos labelled in 0-05 JLLC'I [3H]uridine/ml were developed
104 h after dipping. Developed slides were counter-stained with methyl green/
pyronin and coverslipped. Grains per nucleus were determined by viewing under
oil immersion. Only embryos with cell numbers between 8 and 16 were used to
quantitate RNA synthesis so as to minimize cleavage stage-dependent variability in RNA synthesis, and so as to maximize the comparability of experimental embryos with control embryos. Correction for background levels of
radioactivity were made by counting the number of grains per nucleus for
embryos that were fixed without being incubated in [3H]uridine.
RESULTS
Timing of blastocyst formation
Intact diploid embryos with zonae pellucidae (w/Z-2N) had a t% for cavitation
of 97 h post-hCG, whereas, zona-free diploid embryos (ZF-2N) cavitated about
7 h later (see Table 1, Fig. 2a-c). In fact, after the delay in cavitation accompanying removal of the zona pellucida, further manipulations of the embryos, whether
to reduce cell number, induce tetraploidy, or both, did not result in a statistically
significant change in the timing of cavitation. Regardless of the means used to
induce tetraploidy and regardless of whether or not cell number was restored to
normal, all classes of tetraploid embryos cavitated at the same time as did ZF2N embryos (i.e. about 104 h post-hCG; see Fig. 3). In addition, in the case of
PEG-treated embryos which did not fuse (i.e. PEG-2N), such embryos also had
a t± for cavitation of around 104 h post-hCG (data not shown).
4-2
96
M. A. EGLITIS AND L. M. WILEY
PEG tetraploids
100 T
(a)
3
._
Quartets
.D
Doublets
.*
Singlets
50
103 h '
'S
lOOh
..-•'"
/.-'
..-y\oi
h
.--'*"
.~--
8
89
92
95
98
101 1 0 4 107 110
Hours post hCG
113
116
119
122
Cytochalasin B tetraploids
100,
Restored-whole
Half
73
|
106 h ,'
5 < H
..--
t
102 h
89
92
95
98
101 1 0 4 107 1 1 0 1 1 3 1 1 6 1 1 9 1 2 2
Hours post hCG
Fig. 2. For legend see opposite.
Timing of antigen appearance
At 90 h post-hCG, none of the embryos expressed any antigen on their cell
surfaces, as detected by IIF (Fig. 4). However, at both 94 and 101 h post-hCG,
PEG-4N, w/Z-2N and ZF-2N embryos expressed this embryonic antigen on
their cell surfaces. The antigen was no longer expressed on the trophectoderm of
blastocysts tested by IIF 115 h post-hCG. Thus, antigen expression on tetra-
Development of tetraphid mouse embryos
97
Control diploids
100,
_^
With zonae
.—
Zona-free
...
Half
^
Quarter
(c)
'
• .-•"
3 so -I
to
o
89
92
95
98
101 1 0 4 1 0 7 1 1 0 1 1 3 1 1 6 1 1 9 1 2 2
Hours post hCG
Fig. 2. Timing of cavitation. Embryos were monitored by phase contrast microscopy
for the presence or absence of a blastocoele.
number of embryos with a blastocoele.
% cavitated =
total number of embryos observed
/£ (time at which \ the final maximum number of embryos had cavitated) was
determined by calculating the linear regression for the times at which the number of
cavitated embryos was increasing, then calculating t using the value of \ ' % cavitated'
that was reached at the end of the culture period. Arrow on graphs point to the t\.
(a) PEG Tetraploids: O—O, with cell number restored to whole control levels
(quartets); D---D, with cell number equivalent to i controls (doublets); * — , *
with cell number equivalent to i controls (singlets), (b) Cytochalasin B Tetraploids:
© - - © , with cell number restored to whole control levels (restored-whole); ®---B,
with cell number equivalent to i controls (half), (c) Control Diploids: *—*,
whole diploid embryos with intact zonae pellucidae (with zonae); # — # , while
diploid embryos with zonae pellucidae removed at the 2-cell stage with 0-5 %
Pronase (zona-free); • - - - • , zona-free diploid embryos with % normal cell number
(half); •
*, zona-free diploid embryos with i normal cell number (quarter).
ploid embryos was neither accelerated delayed, nor prolonged when compared
with the controls.
Malate dehydrogenase activity in tetraploid embryos
Because larger numbers of cytB-induced tetraploid embryos could be obtained
these were used in a pilot study on the effect of tetraploidy on MDH activity
(Table 2). In control 2N embryos, between the morula and blastocyst stage,
MDH activity increased 16-2 %, from 4-939 x 10"10 to 5-740 x 10"10 moles NAD
reduced/h/embryo, respectively. In cytB-4N embryos, the increase was 12-3 %
while in |cytB-4N embryos the increase was 23-7 %. In the case of £cytB-4N
98
M. A. EGLITIS AND L. M. WILEY
","*&
o
Development of tetraploid mouse embryos
99
embryos, the percent increase in MDH activity between the morula and blastocyst stage was the same whether such embryos were grown with or without
zonae. In addition, the actual MDH activity of |cytB-4N embryos at each stage
was also approximately the same regardless of whether or not the zona pellucida
was present. Therefore, in the ratios reported below, MDH activities of |cytB4N embryos were determined by pooling values of |w/Z- and |ZF-cytB-4N
embryos.
The MDH activity of cytB-4N morulae (90 h post-hCG) was 1-64 times that
of |cytB-4N morulae. When compared to w/Z-2N embryos, cytB-4N embryos
had 1-54 times the 2N level of enzyme activity. Although the ratio of cytB-4N:
£cytB-4N differs from 2-0 with only borderline significance (Z = 1-544,0-2 > P
> 0-1), the ratio of cytB-4N: w/Z-2N is significantly different from 2-0 (Z =
2-995, P < 0-05). The MDH activity of £cytB-4N morulae was 0-94 times that of
w/Z-2N morulae, not differing significantly from a hypothetical value of 1-0.
At the blastocyst stage (120 h p-hCG), the MDH activity of cytB-4N embryos
was 1-49 times that of £cytB-4N embryos. When compared to w/Z-2N blastocysts, cytB-4N blastocysts had 1-46 times the 2N level of enzyme activity. Both
of these values differ significantly from the expected ratio of 2-0:1-0 (Z = 4-094,
P < 0-05; Z = 5-996, P < 0-01, respectively). The MDH activity of £cytB-4N
blastocysts was 0-98 times that of w/Z-2N blastocysts, not differing significantly
from the hypothetical value of 1-0.
Tetraploid embryos obtained by blastomere fusion were then analysed to
determine if PEG-4N embryos had a depression in MDH activity (relative to the
expected doubling compared to 2N controls) similar to the depression observed
in cytB-4N embryos (Table 3). In these experiments, between the morula and
blastocyst stage, MDH activity in control 2N embryo increased 30-5 %, from
7-601 x 10-10 to 9-916 x 10"10 moles NAD reduced/h/embryo, respectively.
Enzyme activities of diploid embryos grown with or without zonae were pooled
because no differences in activity were detected between the two populations. In
Fig. 3. The appearance, development and ploidy of fused-blastomere tetraploid
embryos (a-c) The appearance of control, ZF-2N embryos, including a representative chromosome spread.
(d-f) The appearance of experimental, PEG-4N embryos.
(a, d) The appearance of diploid and tetraploid embryos at the end of the expermental manipulations (about 60 hpost-hCG injection). The tetraploid embryo (d)
is composed of four fused blastomeres combined by PHA aggregation.
(b, e) Diploid and tetraploid blastocysts about 140 h post-hCG. Note the prominent inner cell mass in both diploid and tetraploid embryos. The tetraploid blastocyst developed from a restored four cell form as illustrated in (d) and was photographed 83 h after PEG-induced fusion.
(c,/) Metaphase plates obtained from diploid and tetraploid-embryos. Blastocysts
were fixed 139 h post-hCG, after a 3 h preincubation in 0-2 /*g/ml colcemid to
maximize cells in metaphase, according to a modification (Epstein et al. 1978) of
Tarkowski's (1966) method. Magnification - bar in e applies to a, b, d and e and
represents 50 /im. Bars in c and/represent 1 /^m.
100
M. A. EGLITIS AND L. M. WILEY
I
u
m
CO
<D
Development of tetraploid mouse embryos
101
PEG-4N embryos, MDH activity increased 37-1 % between the morula and
blastocyst stage. Due to a paucity of material, no PEG-2N embryos were
analyzed at the morula stage, although MDH activities of such embryos were
determined at the blastocyst stage.
The enzyme activity of PEG-4N morulae (90 h post-hCG) was 1-22 times that
of control 2N embryos, which significantly differs from the expected ratio of
2-0 (Z = 12-978, P < 0-01). Thirty hours later, at the blastocyst stage, PEG-4N
embryos had 1-29 times the enzyme activity of 2N embryos, and 0-95 times the
enzyme activity of PEG-2N embryos. Both of these values differ significantly
from the expected ratio (Z = 2-729, P < 0-1; Z = 16-454, P < 0-05, respectively).
Although it might have been expected that the doubled genome of tetraploid
embryos would have been accompanied by a doubling in MDH activity relative
to diploid control embryos, these data show that this correlation was not
detected. This result was observed regardless of the means used to induce
tetraploidy. Rather than a 100 % elevation in enzyme activity, in cytB-4N
embryos the MDH activity was elevated by only 50 %, while in PEG-4N
embryos the MDH activity was elevated by only about 20 %. In fact, in comparing PEG-4N with PEG-2N embryos, their enzyme activities at the blastocyst
stage were essentially the same.
If the doubled genome had been accompanied by doubled enzyme activity,
£PEG-4N or £cytB-4N embryos would be expected to have an enzyme activity
equal to that of w/Z-2N embryos. In the case of |cytB-4N embryos, this is what
was observed. If one assumes that £cytB-4N embryos did indeed have half the
cell number of W/Z-2N embryos (see below), then each $cytB-4N embryo
blastomere had about 190 % the enzyme activity of a w/Z-2N embryo blastomere.
Similarly, the MDH activity of cytB-4N embryos was only 1-5 times the activity
of £cytB-4N embryos. Again, if cytB-4N embryos did, indeed, have twice the
cell number of £cytB-4N embryos, then each blastomere of a -|cytB-4N embryo
had about 130 % the enzyme activity of a blastomere of a cytB-4N embryo.
Fig. 4. The expression of a stage-specific embryonic cell surface antigen on diploid
and tetraploid morulae.
ia-d) Control diploid embryos, grown in culture from the 2-cell stage with intact
zonae pellucidae. Zonas mechanically removed for IIF.
(e-h) PEG-4N embryos.
(a, e) 90 h post-hCG, PEG-4N embryos 31 h after PEG-induced fusion.
(b,f) 94 h post-hCG, PEG-4N embryos 35 h after PEG-induced fusion.
(c, g) 101 h post-hCG, PEG-4N embryos 42 h after PEG-induced fusion.
(d, h) 115 h post-hCG, PEG-4N embryos 56 h after PEG-induced fusion. All
photographs taken with 1 -5 min exposures of Kodak Tri-X film. Prints all exposed for
35 sec. In h, bright patches of fluorescence are caused by blastomeres killed during
11F manipulations which filled with fluorescent antibody. Magnification -bar in
// represents 50 fim.
10
CytB-4N/w/Z-2N
=1-54
i CytB-4N/w/Z-2N = 0-94
CytB-4N/i CytB-4N = 1-64
Activity ratios
A A = Measured change in absorbance/min/10 embryos.
Activity = Moles NAD reduced/h/embryo.
S.E. = Standard error.
n = Number of experiments.
000789 ± 0 0048
000481 ±000025
000512 ±000031
7 611 xlO4-640 xlO" 10
4-939 x 10-10
Activity
id) CytB-4N
(b) ±CytB-4N
(c) W/Z-2N
± S.E.
Morulae (90 h p-hCG)
(« = 10)
(n = 9)
(n = 14)
10
8-547 x lO"
5-740 xlO- 10
5-740 xlO" 10
Activity
CytB-4N/w/Z-2N
= 1-46
i CytB-4N/w/Z2-N = 0-98
CytB-4N/i CytB-4N = 1-49
000886 ±000030
000595 ±000032
0-00608 ±000030
± S.E.
Blastocysts (120 h p-hCG)
Table 2. Malate dehydrogenase activity in cytochalasin B-induced tetraploid embryos
(n = 8)
in = 7)
in = 13)
M
r
m
r
a
a
to
o
y
'
9-299 xlO~10 (n = 8)
7-601 x 10"10 (n = 13)
N.D.
PEG-4N/2N controls = 1-22
«
0-00482±000031
0-00394±0-00023
Activity
AA — Measured change in absorbance/min/5 embryos.
Activity = Moles NAE> reduced/h/embryo.
S.E. = Standard error.
n = Number of experiments.
N.D. = Not done.
Activity ratios
(a) PEG-4N
(b) 2N
(c) PEG-2N
+ S.E.
Morulae (90 h p-hCG)
12-752x lO"10
9-916xlO-10
13-447x 10"10
Activity
PEG-4N/PEG-2N
= 0-95
PEG-4N/2N controls = 1-29
000661 ±0.00041
0-00514±0-00030
000697±0-00047
A/1 + S.E.
Blastocysts (120 h p-hCG)
Table 3. Malate dehydrogenase activity in PEG /fusion-induced tetraploid embryos
(« = 7)
(n = 8)
in = 3)
O
•
104
M. A. EGLITIS AND L. M. WILEY
Table 4. Cell numbers per morula*
Diploid controls
(1) With Zonae
(2) Zonae-free
(3) Total Mean
17-73 ±6-36a
14-47 ±4-02
1614±5-49
—
12-73 ±305
PEG-4N
= 30)
in = 34)
in = 64)
in = 26)
* Cell no. determined at 94 h post-hCG.
standard deviation.
n = number of experiments
a
Cell number of PEG-AN embryos
Cell numbers of PEG-4N, w/Z-2N and ZF-2N morulae were determined 94 h
post-hCG (Table 4) to see whether the data from the MDH assays resulted from
cell loss in tetraploid embryos.
PEG-4N morulae averaged 12-73 ±3-05 cells. w/Z-2N morulae at the same
age had 17-73 ± 6-36 cells, while ZF-2N control embryos had 14-47 ± 4-02 cells,
the total average for the control morulae being 16-14 ± 5-49 cells. The difference
in cell number between tetraploid morulae and control morulae was not
statistically significant (t = 3*01, P < 0*01). Even if the apparent 20% reduction
in cell number was significant, it could not account for the full 40 % reduction
from the expected doubling of MDH activity in quartet tetraploid embryos
relative to whole diploid control embryos.
RNA synthesis in tetraploid embryos
Collectively, the results of the MDH assays and cell number determinations
suggested that neither impaired viability nor reduced cell number could adequately
account for the observed depression of MDH activity in PEG-4N embryos as
compared to an expected level of MDH activity twice that of 2N embryos. One
possible alternative explanation could have been that the depression in enzyme
activity resulted from reduced RNA synthesis in the PEG-4N embryos due to
gene dosage compensation. This hypothesis was tested by quantitating levels of
nuclear RNA synthesis in a series of autoradiographic experiments.
The average number of grains overlying a nucleus as measured in each experiment is shown in Table 5. Because the number of grains per nucleus varied
between experiments, for purposes of comparison, the ratio of PEG-4N grains
per nucleus/2N grains per nucleus was determined for each experiment. In
comparing PEG-4N morulae with w/Z-2N morulae (94 h post-hCG, both
groups of embryos having undergone an equivalent number of cell divisions) the
ratio varied between 0-85 and 1-07, the mean being 0-98. In the comparison
between PEG-4N and ZF-2N morulae, the ratio of grains per nucleus was 1-25
and 1-66, the mean being 1-46. Autoradiographic analysis of PEG-2N embryos
was not undertaken because of a paucity of such embryos, and because the
Development of tetraploid mouse embryos
105
Table 5. Tritiated uridine incorporation into RNA ofmorulae (94 h post-hCG)
Grains/Nucleus (±S.E.)
Level of
Radioactive
label (yMCi/ml)
0-5
0-5
005
no. embryos
(average no. cells/embryos)
,
*
*
w/Z-2N
ZF-2N
PEG-4N
38-15±27-85
25-96±4-61
32-33± 10-43]
n =2
n=4
n=6
}
(1400± 1-41) (12-50± 1-29) (ll-50± 105)J
3O-35± 11-28
19-69± 13*33
32-59± 11-51 ]
n=2
n= 3
n= 5
\
(ll-50± 4-95) (12-67± 3-51) (12-20± 2-28)J
11-11 ± 4-98
—
ll-35± 1-42 1
n =2
—
n= 8
[
(1400± 2-83)
—
(13-25± 212) J
PEG-4N
PEG-4N
w/Z-2N
ZF-2N
0-85
1-25
107
1 66
102
—
0-98
1-46
MDH assays showed that PEG-2N embryos were similar to 2N embryos not
exposed to PEG.
Thus, in no case were nuclei of PEG-4N morulae observed to have twice the
number of grains as nuclei of 2N morulae. Although the ratio of synthesis
between PEG-4N and ZF-2N morulae was greater than that between PEG-4N
and w/Z-2N morulae, in neither case was there evidence that the entire supernumerary genome was expressed. The results of these experiments are consistent
with the idea that the MDH activity in PEG-4N embryos could, at least in part,
be accounted for by partial dosage compensation of the supernumerary genome
in the tetraploid nuclei.
DISCUSSION
In this study we found no statistically significant evidence for an effect of
tetraploidy on the timing of preimplantation development in mouse embryos as
measured by two parameters: (1) the time at which embryos developed a blastocoele and (2) the time at which embryos expressed a stage-dependent cell surface
antigen. This lack of difference in timing of development was observed regardless of whether the tetraploid embryos were produced by PEG-mediated
blastomere fusion, or by cytB incubation. Likewise varying cell number of
either 2N- or 4N- embryos did not affect the timing of cavitation.
These results are in accord with earlier observations by Smith & McLaren
(1977) who detected neither a cell number effect in 2N embryos nor an effect of
cytB-induced tetraploidy on the timing of blastocoele formation. It should be
pointed out that in the Smith and McLaren study, their cytB-4N embryos corresponded to our JcytB-4N embryos.
MDH activity, which served as a parameter of embryonic metabolism, was
106
M. A. EGLITIS AND L. M. WILEY
monitored to determine whether the response of embryonic metabolism to tetraploidy might provide an explanation for the timing data. We found that cytB4N embryos had only 1-5 times the MDH activity of 2N embryos, while PEG4N embryos had only 1-2 times the MDH activity of 2N embryos. Thus, MDH
activity, and presumably, overall embryonic metabolism, did not increase by a
factor of two in response to a 2-fold increase in the embryonic genome.
These data on MDH activity might simply be a result of depressed embryo
viability of 4N embryos, with perhaps, PEG reducing embryo viability more
than cytB. However, although there is some difference in MDH activities between
cytB-4N and PEG-4N embryos relative to their respective 2N controls, the
actual MDH activity levels of cytB-4N and PEG-4N embryos are very similar.
In addition, in both types of 2N embryos, the increase in MDH activity as 4N
morulae developed into blastocysts was very similar in magnitude to the increase
in MDH activity of 2N morulae as they developed into blastocysts. Thus, for
both types of 4N embryos, the ratios of enzyme activity (cytB-4N:2N and PEG4N:2N) remained constant, not decreasing between the morula and the blastocyst stage as would be expected if the embryos were progressively deteriorating.
If, then, tetraploidy itself impairs embryo viability and if PEG impairs viability
more so than cytB, then these impairments must have remained constant over
the 30 h period during which MDH activities were measured. A final argument
against these data being a total result of depressed embryo viability are two
observations, namely, (1) cytB-4N and PEG-4N embryos formed blastocysts at
similar frequencies and (2) both types of 4N embryos had similar timing rates
for blastocyst formation.
Another possible explanation for the apparent depressed level of MDH
activity in 4N embryos is that measurements of enzyme activity could have been
made at a point where the activity curve had reached a maximum. Only in the
region of the curve where activity per unit substrate was uniformly increasing
would it be possible to detect a doubling of activity. If measurements were made
in the non-linear region of the activity curve, then, as more embryos were
assayed per sample, activity per embryo would decrease. This, however, was not
the case, since the numbers of PEG-4N embryos per sample varied between 3
and 5 with no change in calculated activity per embryo. The correlation of
activity-to-embryos per sample is 0-419 for morulae (P > 0-1 that activity is
independent of number of embryos) and -0-031 for blastocysts (P > 0-5 that
activity is independent of number of embryos). Finally, our 2N levels of MDH
activity are in good agreement with those previously reported by Brinster (1966),
suggesting that our MDH data are cogent.
Another possible explanation for the MDH data is that 4N embryos had fewer
cells than did 2N embryos. However, the difference in cell number between 4Nand 2N embryos was not statistically significant, and cannot, therefore, account
for the MDH data.
If neither depressed embryo viability, nor errors in enzyme activity measure-
Development of tetraploid mouse embryos
107
ments, nor cell loss can account for the MDH data, could chromosomal loss or
gene dosage compensation explain the observed, less than 2-fold increase in
MDH activity in 4N embryos? Our chromosomal counts showed that putative
4N embryos were truly 4N with no evidence of partial chromosomal loss or
2N/4N mosaicism (these results and Eglitis, 1980). Thus, chromosomal loss is
probably an unlikely reason for the MDH data.
To determine whether gene dosage compensation might account for the MDH
data, RNA synthesis was measured autoradiographically in 2N and 4N embryos. The resulting grain counts indicated that the mean level of RNA synthesis
in PEG-4N embryos was 1 -45 times that of ZF-2N embryos and 0-98 times that of
w/Z-2N embryos. The difference in these two ratios is due to finding that the
level of RNA synthesis in ZF-2N embryos was 35 % lower than that in w/Z-2N
embryos. This finding could, then, be evidence for decreased viability of zonefree embryos in general, including, then, of the PEG-4N embryos whose levels
of RNA synthesis were measured in these experiments. This would mean that the
higher of these two ratios (1-45) is the more meaningful result. However, it is
noteworthy that of all three embryo types (ZF-2N, w/Z-2N and PEG-4N), the
standard error is greatest for ZF-2N embryos. It is possible, then, that the higher
ratio results from a sampling error, particularly since the elevated ratio stems, in
large part, from one experiment.
Regardless of whether the higher or the lower ratio is more accurate, it is clear
that PEG-4N embryos do not synthesize twice as much RNA as do 2N embryos. Although the possibility remains that the RNA data result from decreased embryo viability, for the reasons discussed earlier, reduced embryo
viability probably cannot explain all of the observed MDH and RNA data. These
results are, however, compatible with the intriguing possibility that in PEG-4N
morulae, compensation is made for at least part of the excess genetic material.
Although RNA synthesis in |cytB-4N embryos was not quantitated, comparison of the MDH activities in £cytB-4N embryos with that of cytB-4N embryos
suggests that the excess genome was not as thoroughly compensated for in -|cytB4N embryos as it was in cytB-4N embryos. This possibility is consistent with the
idea that the control of gene activity is sensitive to cell number-related cell-tocell interactions.
In 1977, Smith & McLaren concluded that the control of developmental
timing most likely resided in the ratio of nuclear-to-cytoplasmic volume, or, in
the number of nuclear divisions (DNA replications) that an embryo had undergone. The results here do not contradict either of these possibilities. In both
PEG-4N and cytB-4N embryos, the ratio of nuclear-to-cytoplasmic volume
remains the same as in 2N embryos of the same age. In blastomere fusion, as
nuclear volume increases with tetraploidy, so does the cytoplasmic volume.
Similarly, nuclear volume in cytB-4N embryos doubles, and, since, cell division
is blocked during cytB treatment, cytoplasmic volume also doubles. Tetraploid
embryos probably undergo the normal number of nuclear divisions since neither
108
M. A. EGLITIS AND L. M. WILEY
cell numbers nor ploidy are perturbed past the morula stage (Snow, 1973;
Eglitis, 1980).
Because of the evidence for some degree of gene-dosage compensation in 4N
embryos, our results do not permit a conclusion to be reached on the question
of how great a role the embryonic genome actually has in developmental timing.
However, these results do suggest that the nucleus is under tight control that may
involve nuclear-cytoplasmic interactions sensitive to cell number.
We would like to thank Dr Irwin R. Konigsberg for some useful suggestions, and Dr E. E.
Oliphant for his helpful criticisms during the preparation of the manuscript. Dr Patricia
Rodier assisted with the statistical analyses, while Dr Donald A. Keefer helped with the autoradiographic study, to which Dr S. K. Lau lent his technical expertise. This report was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy with
the Department of Anatomy, University of Virginia. This work was supported by an NIH
grant to L.M.W., NICHHD 1-RO1-11788. M.A.E. had additional support from an NIH
predoctoral training grant.
REFERENCES
ALEXANDRE, H. (1979).
The utilization of an inhibitor of spermidine and spermine synthesis as
a tool for the study of the determination of cavitation in the preimplantation mouse embryo.
J. Embryol. exp. Morph. 53, 145-162.
BIGGERS, J. D., WHITTEN, W. K. & WHITTINGHAM, D. G. (1971). The culture of mouse
embryos in vitro. In Methods of Mammalian Embryology (ed. J. C. Daniel, jr), pp. 86-116.
San Fransisco: W. H. Freeman.
BRAUDE, P. B. (1979). Time-dependant effects of a-amanitin on blastocyst formation in the
mouse. J. Embryol. exp. Morph. 52, 193-202.
BRINSTER, R. L. (1966). Malic dehydrogenase activity in the preimplantation mouse embryo.
Expl Cell. Res. 43, 131-135.
CHAPMAN, V. M., WHITTEN, W. K. & RUDDLE, F. H. (1971). Expression of paternal glucose
phosphate isomerase-1 (Gpi-1) in preimplantation mouse embryos. Devi. Biol. 26,153-158.
EGLITIS, M. A. (1980). Formation of tetraploid mouse blastocysts after blastomere fusion
with polyethylene glycol. J. exp. Zool. 213, 309-313.
EPSTEIN, C. J., SMITH, S., TRAVIS, B. & TUCKER, G. (1978). Both X-chromosomes function
before X-chromosome inactivation in female mouse embryos. Nature 21A, 500-503.
KNOWLAND, J. & GRAHAM, C. (1972). RNA synthesis at the two-cell stage of mouse development. /. Embryol. exp. Morph. 27, 167-176.
MINTZ, B. (1962). Experimental study of the developing mammalian egg: Removal of the
zona pellucida. Science 138 594-595.
MINTZ, B., GEARHART, J. D. & GUYMONT, A. O. (1973). Phytohemagglutinin-mediated
blastomere aggregation and development of allophenic mice. Devi. Biol. 31, 195-199.
SMITH, R. & MCLAREN, A. (1977). Factors affecting the time of formation of the mouse
blastocoele. /. Embryol. exp. Morph. 41, 79-92.
SNOW, M. H. L. (1973). Tetraploid mouse embryos produced by cytochalasin B during
cleavage. Nature 244, 513-514.
SPINDLE, A. I. & GOLDSTEIN, L. S. (1975). Induced ovulation in mature mice and developmental capacity of embryos in vitro. J. Reprod. Fert. 44, 113-116.
SURANI, M. A. H., BARTON, S. C. & BURLING, A. (1980). Differentiation of 2-cell and 8-cell
mouse embryos arrested by cytoskeletal inhibitors. Expl Cell Res. 125, 275-286.
TARKOWSKI, A. K. (1966). An air-drying method for chromosomal preparations from mouse
eggs. Cytogenetics 5, 394-400.
WUDL, L. & Chapman, V. (1976). The expression of /ft-glucuronidase during preimplantation
development of mouse embryos. Devi Biol. 48, 104-109.
(Received 18 September 1980, revised 19 May 1981)