/. Embryo/, exp. Morph. Vol. 46, pp. 53-64, 1978
Printed in Great Britain © Company of Biologists Limited 1978
53
X-chromosome activity in preimplantation mouse
embryos from XX and XO mothers
By MARILYN MONK 1 AND MARY HARPER 1
From the MRC Mammalian Development Unit, Wolfson House, London
SUMMARY
Embryos from XO female mice begin development with half the activity levels of an
enzyme (HPRT) coded for by a gene on the X chromosome, compared with embryos from
XX females. Groups of unfertilized eggs and individual embryos at the 8-cell, morula and
blastocyst stages were assayed for HPRT activity. An autosomally coded enzyme (APRT)
was assayed simultaneously in the same reaction mix as a control. There is a substantial
increase in HPRT activity by the 8-cell stage. However, the mean activity of HPRT in
embryos of XO mothers remains half that in embryos of XX mothers. This suggests a significant maternally inherited component of HPRT activity in 8-cell embryos. By the 9- to 16-cell
morula stage the HPRT activities in the two groups of embryos become similar due, presumably, to a transition to embryo-coded activity; HPRT activities in individual morulae
from XX mothers show a bimodal distribution consistent with the hypothesis that both Xchromosomes are active in XX embryos at this stage.
INTRODUCTION
Previous work (Monk & Kathuria, 1977) has shown that JfXand XY embryos
appear to be equivalent with respect to X-linked HPRT (hypoxanthine phosphoribosyl transferase, E.C. 2.4.2.8) activity at the early 8-cell and the blastocyst
stages. The distribution of HPRT activities for large numbers of individual
embryos did not correspond to two populations (XX and XY) differing by
a factor of two, which might be expected if XX embryos had twice the HPRT
activity of XY embryos. We concluded that some form of dosage compensation
for the ^-linked HPRT activity was operating at these developmental stages.
Although there is evidence that HPRT activity is embryo-coded in morulae
and blastocysts on the fourth day of pregnancy (Epstein, 1972), it remained
possible that the activity at the 8-cell stage was maternally derived.
In this work we have extended Epstein's earlier studies (Epstein, 1972) to an
investigation of levels of HPRT activity in single embryos throughout preimplantation development. This enzyme is coded by the X chromosome, and
because both X chromosomes are active during oogenesis (Epstein, 1969, 1972;
Kozak, McLean & Eicher, 1974) embryos of XO mothers begin development
with half as much HPRT as do embryos of XX mothers. The time at which
1
Authors' address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London NW1 2HE, U.K.
r
54
M. MONK AND M. HARPER
activities in the two groups of embryos become similar presumably reflects
the onset of expression of the embryonic HPRT gene. We show here that there
is a considerable increase in HPRT activity up to the 8-cell stage although the
activity levels in XO-derived embryos remain half those in AX-derived embryos.
As a control we show that activities of an autosomally-coded enzyme, APRT
(adenine phospho-ribosyl transferase, E.C. 2.4.2.7), remain similar in the two
groups of embryos at each stage, while also rising with advancing development.
Beyond the 8-cell stage HPRT levels become the same in XX- and ZO-derived
embryos as the embryo-coded HPRT activity predominates. The distribution
of HPRT activities in individual 9- to 16-cell embryos from XX mothers is
bimodal with a twofold separation of the modes. This result suggests that both
X chromosomes are active in female XX embryos prior to implantation.
MATERIALS AND METHODS
Mouse embryos were obtained from randomly bred MF1 females (Olac 1976
Ltd) or from XX and XO females (genotypes Ta/ + and + / O ; kindly provided
by Mary Lyon, MRC Radiobiology Unit, Harwell). The mice were superovulated by intraperitoneal injection of 5 i.u. of PMS (pregnant mare serum)
and, 48 h later, 5 i.u. of HCG (human chorionic gonadotrophin) and then
immediately caged with MF1 males. Eggs were isolated from the oviducts the
morning following mating, 2-cell, and 8-cell and 9- to 16-cell embryos from the
oviducts on the second and third day of pregnancy respectively, and early and
late blastocysts from the uterus on the fourth and fifth day of pregnancy
respectively. The medium used for collection of eggs and embryos (PB1) and
the methods of isolation of embryos are described in Monk & Ansell (1976).
The enzymes HPRT and APRT in embryo extracts were simultaneously
assayed in the same reaction mix (McBurney & Adamson, 1976; Monk &
Kathuria, 1977). XX- or ZO-derived eggs, 8-cell embryos and 9- to 16-cell
morulae and blastocysts were transferred to 10 /U Drummond caps, either
singly or in groups of five to ten, in less than 5 /A of medium. The microcaps
were sealed and extracts prepared by freeze-thawing in liquid nitrogen three
times, followed by centrifugation for 10 min. The supernatant from each embryo
extract was added to a 50 /A reaction mixture containing sodium phosphate
buffer (35 mM, pH 7-4), magnesium chloride (5 mni), phospho-ribosyl pyrophosphate (2 mM), [3H]guanine (10 /*M, specific activity 84 mCi/mM) and [14C]adenine (10/AM, specific activity 58 or 61 mCi/mM). The reactions were carried
out at 37 °C for times ranging between 2 and 4 h and stopped by the addition
of 1 ml volumes of cold lanthanum chloride (0-1 M) containing adenine and
guanine (1 mM). The linearity of the reactions with times of incubation for
the embryos examined had previously been established (Monk & Kathuria,
1977). Corrections for spill-over of [3H] counts into the [14C] channel, and vice
versa, were determined in reactions containing embryo extract and [3H] or [14C]
alone respectively. The level of counts ranged from three times ([14C] counts
X-Chromosome activity in preimplantation mouse embryos
55
3
for 8-cell embryos) to ten times ([ H] counts for blastocysts) the counts in
replicate reproducible blank samples containing 5 jil of supporting medium but
no embryo extract. Counting efficiencies for [3H] and [14C] were determined
by spotting 2/d of reaction mixture directly onto filters. Some day-to-day
variability was unavoidable, e.g. the calculated enzyme activities shown in
Figs. 1, 4 and 5 are relatively low, but the data in each experiment are internally
consistent and this variability does not affect the interpretation of the results.
For single embryo assays increased accuracy is achieved by the expression
of the results as ratios of HPRT:APRT activities where both enzymes are
measured simultaneously in the same reaction mix. This eliminates variance
due to variable recovery of embryo extracts.
RESULTS
Throughout these experiments we have assayed both HPRT, coded by the
X chromosome, and as a control, APRT which is coded autosomally. Fig. 1
shows the increase in activities of HPRT and APRT during preimplantation
development. The two enzymes were measured simultaneously in a given batch
of embryos at a particular stage of development. Essentially similar results were
previously published by Epstein (1970) who used high voltage paper electrophoresis to measure each enzyme separately in extracts of embryos at comparable stages.
Because both X chromosomes are active during oogenesis, embryos from
XX mothers would have twice the levels of HPRT compared with embryos
from XO mothers for as long as the activity observed was maternal in origin.
Fig. 2 shows the twofold difference in HPRT activities in unfertilized eggs from
XO and AX females, thus confirming the results of Epstein (1972). The ratios of
activities of HPRT: APRT in the XO and XX eggs are different by a factor of
three due to rather higher APRT activities in the XO eggs in these particular
experiments.
The activities of HPRT, APRT, and their ratios in single 8-cell embryos from
XX and XO (hatched squares) mothers are shown in Fig. 3. Although the
autosomally coded APRT activities are similar in both groups of embryos, the
HPRT activities, and hence the HPRT:APRT ratios, are approximately twofold lower for XO embryos, thus reflecting the maternal origin (XX or XO) of
the embryo (Fig. 2). In addition the HPRT activities in the XY-derived 8-cell
embryos in Fig. 3 are consistent with a bimodal distribution (modes approximately 1-5:1) which might indicate the separation of XX and XY embryos due
to an effect of X-chromosome dosage on embryo-coded HPRT activity. Similar
bimodal (approximately 1-5:1) distributions were obtained in four other
experiments with AX-derived 8-cell embryos.
To determine when embryo-coded activity began to obliterate maternal
differences we examined slightly older embryos, namely morulae at the 9- to
16-cell stage. These were compacted and an attempt was made to include only
56
M. MONK AND M. HARPER
12 -
11 10 -
- 40
9-
- 36
|
i s
- 32 2
x
o
- 28 b
X)
p
- 24 - 5
- 20
X
I
4
o
-16 H
3
- 12 <
9
- 8 4
1 -
- 4
\
I
I
I
10
20
30
40
t
1 cell
I
I
1 I
2 cell
I
I
I
50 60
70 80 90
Hours after mating
8 cell
1
I
100 110
i 1
Blastocyst Late blastocyst
Fig. 1. Activities of HPRT and APRT in developing MF1 embryos. Fertilized eggs,
2-cell embryos, 8-cell embryos and morulae, and early and late blastocysts were
isolated on the first, second, third, fourth and fifth days of pregnancy respectively.
Extracts from batches of five to ten embryos were assayed for HPRT and APRT.
Assays were performed for 3 h as described in Materials and Methods. Results are
plotted against the number of hours after mating, assuming mating occurs at
midnight.
those where the outline of the morula showed a fourth cleavage division had
occurred. Figure 4 shows that XO-derived morulae (hatched squares) now
have the same HPRT, APRT and HPRT: APRT ratios as JOT-derived morulae.
We conclude that HPRT activity is embryo-coded in 9- to 16-cell morulae
isolated late on the third day of pregnancy. An analysis of a large number of
individual 9- to 16-cell morulae derived from IX-mothers shows a clear bimodal
distribution, with an approximate twofold separation of the modes, for HPRT
and HPRT: APRT ratios (Fig. 5). The distribution of values for the autosomal
enzyme, APRT, is unimodal. The results suggest two populations of embryos,
JOT and XY, differing by a factor of two with respect to X-chromosome activity.
X-Chromosome activity in preimplantation mouse embryos
xx
XO
0-15 0-2 0-25 0-3 0-35 0-4 0-45
HPRT(p-mole/h/egg)
XX
„ R
XO
14
16 18 20 22 24 26 28
APRT (p-mole/h/egg x 102)
XX
\ \
\ \
xo
0-4 0-8 1-2 1-6 20 2-4 2-8 30
Ratio HPRT/APRT
Fig. 2. Activities of HPRT, APRT, and ratios of activities HPRT: APRT in batches
of eggs (five to ten eggs per batch) from XX and XO (hatched squares) females.
Each square represents a separate batch of eggs. The mice were superovulated and
unfertilized eggs collected at approximately 21 h following HCG injection. Assays
were performed for 4 h. Mean activities (p-mole per hour per egg± S.E.), AT eggs:
HPRT 0-38±001, APRT 0-16±0-005, HPRT/APRT 2-4±007; XO eggs: HPRT
017±001, APRT 0-21 ±001, HPRT/APRT 0-8±006.
57
58
M. MONK AND M. HARPER
XX
XO
2
3
4
5
6 7 8 9
H P R T (p-mole,h)
10
11
12
14
XX
n
U.
XO
Kl N
10
15 20
25 30 35 40 45
APRT (p-mole h x 102)
XX
XO
JSL
6
12
18
24
30 36 42 48 54 60
Ratio HPRT/APRT
Fig. 3. Activities of HPRT, APRT, and ratio of activities HPRT: APRT in single
8-cell embryos isolated from XX and XO (hatched squares) females on the third day
of pregnancy at 64 and 68 h, respectively, after HCG injection. Each square
represents a value obtained for a separate embryo. The results from two experiments
were pooled. Assays were performed for 4 h. Mean activities (p-mole per hour per
embryo±S.E.), XX embryos: HPRT 7-5 + 0-4, APRT 0-22±001, HPRT/APRT
35-4+1-4; XO embryos: HPRT 4-0±0-3, APRT 0-22±001, HPRT/APRT
18-9±l-2.
X-Chromosome activity in preimplantation mouse embryos
XX
-
n
1\
s\
s
1 1 1 1 1
XO
MMMMN ,
2
4
6
8
10 12
HPRT(p-mole/h)
XX
XO
s
6
6
12sKJ8s 24
96
30 36 42 48
APRT(p-mole/h.x 102)
12 .18 24 30 36 42 48 54
Ratio HPRT/APRT
60
Fig. 4. Activities of HPRT, APRT, and ratios of activities HPRT:APRT in single
9-16 cell morulae isolated from XX and XO (hatched squares) females at 70 and
76 h, respectively, after HCG injection. Assays were performed for 4h. Mean
activities (p-mole per hour per embryo ± S.E.), XX embryos: HPRT 62 ± 0-6, APRT
016±001, HPRT/APRT 37-8±29; XO embryos: HPRT 71 ±13, APRT
0-25 ± 006, HPRT/APRT 32-3 ± 3-3.
59
60
M. MONK AND M. HARPER
Fhr
0-5
2-5
10
-Fh-m
30
1-5
2-0
2-5
HPRT (p-mole/h/embryo)
7-5
10
12-5
APRT (p-mole/h/embryo x 102)
12
16
15
17-5
20
24
Ratio HPRT/APRT
Fig. 5. Activities of HPRT, APRT, and ratios of activities HPRT/APRT in single
9-16 cell morulae isolated from MFl females at 74 h after HCG injection. Assays
were performed for 4 h. Mean activities (p-mole per hour per embryo ± S.E.) HPRT
1-51 ±007, APRT 011 ±0003, HPRT/APRT 13-06 + 0-48.
X-Chromosome activity in preimplantation mouse embryos
xx
XO
• N
4
8
12
16
20 24 28 32 36 40 44 48 52
HPRT(p-mole/h)
XX
r
XO
\ \
\ \
r\l\)
• . f\3
\
10 20 30 40 50 60 70 80 90 100 110
APRT(p-mole/hx 102)
\I\T\
XX
J_L
m n
XO
JSL
10 20 30 40 50 60 70 80 90 100
Ratio HPRT/APRT
180
Fig. 6. Activities of HPRT, APRT, and ratios of activities HPRT: APRT in single
blastocyst isolated from AX and XO (hatched squares) females on the fourth day of
pregnancy at 97 and 99 h, respectively, after HCG injection. Assays were performed for 3 h Mean activities (p-mole per hour per embryo ± S.E.), XX embryos:
HPRT 290 ± 1-7, APRT 0-48 ± 001, HPRT/APRT 59-6 + 2-7; XO embryos: HPRT
28-4 + 2-8, APRT 0-57±006, HPRT/APRT 58-7±9-7.
EMB 46
61
62
M. MONK AND M. HARPER
Blastocysts isolated from XO and XX females on the next (4th) day of
pregnancy (Fig. 6) also show equivalent levels of HPRT and APRT activities,
but now the separation of presumptive XX and XY embryos is less than twofold.
DISCUSSION
Epstein (1972) has previously studied the activities of HPRT in eggs and 2-cell
stage embryos, and in morulae and blastocysts on the 4th day of pregnancy,
in batches of embryos issuing from XX and XO mothers. Eggs and 2-cell stage
XX-dtnvQd embryos had twice as much HPRT activity as those from XO
mothers, thus reflecting the X chromosome dosage during oogenesis. There was
little, if any, increase in activity from the egg to the 2-cell stage in embryos from
either XX or XO mothers. Epstein did not report results for embryos taken on
the third day of pregnancy, though on the fourth day of pregnancy the approximate equivalence of HPRT activities in batches of embryos from XO and XX
mothers led him to postulate that, at this stage, HPRT activity was embryocoded.
Embryos from XX and XO mothers will have different sex chromosome constitutions (XX and XY, and XX, XY, XO and YO, respectively). The YO
embryos are thought to be arrested at the 8-cell stage (Burgoyne & Biggers,
1976). Theoretically, if both X chromosomes had been active in generating the
activity observed in groups of blastocysts (Epstein, 1972), then the XO-derived
embryos should show a predictable fraction (0-89 or higher) of the HPRT
activity of the XJf-derived embryos. This fraction is derived from the expectation
of four active X chromosomes in three embryos (XX, XO and XY) compared
with three active X chromosomes in two embryos (XX and XY). The value
would be greater than 0-89 since the proportion of XO embryos from the XO
mothers is less than one third (Kaufman, 1972). If X inactivation had occurred,
HPRT activity in groups of blastocysts from XX and XO mothers should be
equivalent. Epstein could not deduce from his data whether both X chromosomes
were active in preimplantation development.
The application of the above argument to the data in Fig. 3 indicates that
HPRT activity in the 8-cell embryos analysed is unlikely to be totally embryocoded. If this were so we would expect the ratio of the mean HPRT activities,
v
n
w
+
,
-t-lXO
XO.XX, to be 0-67 I
+ OY+XX+XY
TTT?—^
4/4\
= ^
...
or higher if XO plus OY
embryos are present to less than 50 per cent of the litter; or 0-75 (^75) or higher
\Lj 1]
if only one X chromosome is active in XX embryos up to the 8-cell stage.
Statistical analysis shows that the ratio of the mean HPRT activities, XO to
XX, in Fig. 3 (0-53) is significantly different from 0-67 (P < 0-01).
On the other hand the indications of an approximately 1-5-fold separation
of two populations with respect to HPRT activity in the AX-derived 8-cell
embryos in Fig. 3 would suggest that some embryo-coded activity is already
X-Chromosome activity in preimplantation mouse embryos
63
present. The data presented in Fig. 3 can be most easily interpreted as resulting
from a mixture of approximately equal amounts of maternally inherited and
embryo-coded HPRT activity. We previously reported a unimodal distribution
of HPRT activities for 8-cell embryos (Monk & Kathuria, 1977). When reexamined at the level of single litters of embryos this earlier data is also consistent with 1-5 fold separation of two populations of embryos. Similar results
have been obtained by Kratzer & Gartler (pers. comm.). Since there is a 10- to
20-fold increase in HPRT activity by the 8-cell stage it seems highly likely that
some increase has occurred in maternally derived enzyme activity. These
results may therefore represent the first evidence in mammalian embryos of
active stable maternal messenger RNA functional up to the fourth cleavage in
preimplantation development, although the gradual activation of pre-existing
maternal precursor protein for HPRT activity cannot be excluded.
An alternative explanation for the lack of a twofold separation of embryos
at the early 8-cell stage might be that the onset of expression of the maternally
derived HPRT gene occurs earlier than that on the paternally derived X chromosome. This appears unlikely in view of the fact that there is no evidence in Fig. 3
of XX and XY embryos from XO mothers with equivalent activity to those
from XX mothers. However, embryos isolated from XO mothers may be delayed
in development (Burgoyne & Biggers, 1976). For the experiment shown in
Fig. 3 we were careful to select all embryos at the 8-cell stage, and embryos
from XO mothers were collected some hours later than those from XX mothers.
Also a recent careful analysis of HPRT and APRT activities throughout
development with several time points on the third day of pregnancy has shown
that HPRT: APRT ratios do not vary by as much as a factor of two from early
to late 8-cell stages (Harper & Monk, unpublished). There is no sign of a
developmental lag of XO-derived embryos at the morula stage (Fig. 4).
In morulae, where the HPRT activity is embryo-coded, the bimodal distribution with respect to HPRT activities strongly suggests that both X chromosomes are active in female embryos. A similar conclusion has been advanced
by Adler et al. (1977), and further evidence has been obtained by Kratzer &
Gartler (pers. comm.) and Epstein (pers. comm.). In blastocysts the separation
of presumptive AX and XY embryos is less than twofold. This result is consistent
with the earlier observation (Monk & Kathuria, 1977) that X-inactivation has
occurred in most, if not all, of the cells of the blastocyst.
We thank Paul Kratzer, John West and Anne McLaren for advice in the preparation of
this manuscript.
REFERENCES
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BURGOYNE, P. S. & Biggers, J. D. (1976). The consequences of X-dosage deficiency in the
germ line: impaired development in vitro of pre-implantation embryos from XO mice.
Devi Biol. 51, 109-117.
ADLER,
5-2
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M. MONK AND M. HARPER
C. J. (1969). Mammalian oocytes: X chromosome activity. Science, N.Y. 163,
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EPSTEIN, C. J. (1970). Phosphoribosyl transferase activity during early mammalian development. /. biol. Chem. 245, 3289-3294.
EPSTEIN, C. J. (1972). Expression of the mammalian X chromosome before and after fertilisation. Science, N.Y. 175, 1467-1468.
KAUFMAN, M. H. (1972). Non-random segregation during mammalian oogenesis. Nature,
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MCBURNEY, M. W. & ADAMSON, E. D. (1976). Studies on the activity of the X chromosome
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EPSTEIN,
(Received 10 November 1977, revised 21 April 1978)