773
Development 102. 773-779 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Diploid mouse embryos constructed at the late 2-cell stage from haploid
parthenotes and androgenotes can develop to term
J. BARRA and J.-P. RENARD
Vmti de Gintuque des Mammiferes, InstituI Pasteur, 25 rue du Docteur Roux, 75015 Paris, France
Summary
Male and female gamete nuclei are required to ensure
the full-term development of the mouse embryo.
Differential expression of the two genomes has been
proposed as the basis for this requirement. In order to
investigate whether some interactions between the
paternal and the maternal genomes are essential
before or at the time of the activation of the embryonic
genome, we have constructed diploid embryos from
haploid parthenotes and androgenotes at the late 2-cell
stage. These embryos developed to term into normal
offsprings. This shows that the male and the female
genomes can be activated separately and are still able
to ensure complete development when put together in
cytoplasm synchronized with the nuclei. These experiments also show that the egg cytoplasm does not need
any male contribution before the late 2-cell stage.
Introduction
during mouse development (Surani et al. 1984;
•McGrath & Solter, 1984a).
It is not known at which stage the differences
between paternal and maternal genomes are first
expressed during embryonic development. In the
mouse, embryonic genes are active as early as the
early 2-cell stage (Flach et al. 1982; Bensaude et al.
1983; Bolton et al. 1984). One can ask whether, in
order to ensure full development, some interactions
between the two genomes, directly or via the cytoplasm, must take place prior to this stage.
In the present paper, we show that a late 2-cell
haploid male nucleus can be introduced into a late 2cell haploid parthenogenetic blastomere to form a
diploid embryo that will develop normally to term.
Over the past three years, nuclear transfer experiments have firmly established that the participation
of both male and female gamete nuclei is essential for
full-term development of mouse embryos (Surani et
al. 1984; McGrath & Solter, 1984a; Mann & LovellBadge, 1984).
Genetic observations have revealed sex-linked differential expression of the embryonic genome, such
as the preferential inactivation of the paternal X
chromosome in extraembryonic tissues (Takagi &
Sasaki, 1975; Harper et al. 1982) or the expression of
the T-hairpin mutation (Johnson, 1974; McGrath &
Solter, 19846). Cattanach and colleagues, using various rearrangements of the chromosomes, have shown
that while the sex origin of most chromosomes seems
unimportant, some pairs of homologous chromosomes are lethal when provided by a single parent.
They have also shown that some chromosomal regions express abnormal and opposite phenotypes
according to the sex origin (Cattanach & Kirk, 1985;
Cattanach, 1986). Differential imprinting of the male
and female genomes (Crouse, 1960) has been proposed to account for their differential expression
Key words: nuclear transfer, electrofusion, parthenote,
androgenote, imprinting, mouse embryo.
Materials and methods
Strains of mice
Females C57BL/7XCBA (agouti; henceforth called F,),
homozygous for the enzyme glucose phosphate isomerase
(GPI-lb/GPl-lb) were used at 3-4 weeks of age as donors
of oocytes. Females BALB/cxSJL (albinos, henceforth
called F,BS), homozygous for the glucose phosphate isomerase (GPI-la/GPI-la) were mated at 3 weeks of age with
774
J. BarraandJ.-P. Renard
BALB/c males (GPI-la/GPI-la) to obtain eggs. Mice were
superovulated by injecting 5i.u. of pregnant mare's serum
gonadotrophin (PMS) followed 44—48 h later by an injection of 5 i.u. of human chorionic gonadotrophin (HCG).
Recipient pseudopregnant mice were F] females mated
with vasectomized F) males. The morning that a vaginal
plug was detected was designated as day 1.
Haploid embryo preparation and culture
Parthenotes
Unfertilized eggs surrounded by the cumulus cells were
recovered 17-20 h after the injection of HCG. Eggs were
activated by culture for 5min 30 s at room temperature in
Whitten medium (Whitten, 1971) supplemented with 6%
ethanol (Cuthbertson, 1983), washed several times and
kept in culture in the same medium under paraffin oil at
37°Cin7% CO 2 in air. After 4-5 h of culture, the cumulus
cells were dispersed by incubation for 5min at 37°C in
0-1 % hyaluronidase in Whitten medium. The haploid eggs
that had extruded the second polar body and had a visible
pronucleus were selected (Fig. 1A) and kept in culture.
Androgenotes
Fertilized eggs were obtained at the same time. Enucleation
of the female pronucleus was carried out on a Leitz
micromanipulator as described by McGrath & Solter (1983)
(Fig. IB). The eggs were placed in phosphate-buffered
medium (PB1) (Whittingham & Wales, 1969) containing
cytochalasin D (0-5/igml"1) and Nocodazole (0-01 /igml"1)
for 30-45 min before the microsurgery was performed. The
haploid androgenotes were washed 5-6 times and cultured
as described above.
Reconstitution of diploid embryos
2-cell-stage haploid parthenogenetic embryos were used as
recipients and 2-cell-stage haploid androgenotes were used
as donors. The donor embryo was incubated for at least
30 min in Whitten medium containing cytochalasin and
Nocodazol. The nucleus of one of the blastomeres was
sucked into a small cytoplasmic vesicle (karyoplast) and
introduced into the perivitelline space underneath the zona
pellucida of the recipient parthenogenetic haploid embryo,
a blastomere of which had been previously enucleated
(Fig. 1C,D). Karyoplast/cell fusion was mediated either by
inactivated Sendai virus particles (McGrath & Solter, 1983)
or by electrofusion (Fig. IE, see below).
The reconstituted embryos were kept in culture for 5-7 h
after fusion. All of the embryos where both nuclei were
visible in the same cell (Fig. IF), were transferred to one of
the oviducts of a pseudopregnant mouse at day 1 while
nonmicromanipulated diploid embryos were transferred to
the contralateral oviduct.
Electrofusion technique
Electrofusion was performed under a Leitz dissection
microscope by modifying the original technique proposed
for mouse embryos by Kubiak & Tarkowski (1985). The
electrodes, 0-5 mm long, made from 0-1 mm diameter
platinum wire, were fastened at a distance of 0-5 mm on the
surface of the cover of a Petri dish. They were connected to
a stimulator (GRASS S44) that generated square electric
pulses. An oscilloscope (METRIX OX 712D) was used to
control the parameters of the electric pulses.
The fusion was performed in a drop (70 //I) of Whitten or
PB1 medium. One or two embryos at a time were placed
between the electrodes so that the membranes to be fused
were parallel to the electrodes (Fig. IE). A single pulse of
40-50 V was applied for 480/«. The embryos were washed
and kept in Whitten medium at 37°C. They were observed
every 15min. Fusion was completed after 30-60min.
GPI determination
GPI-1 isozymes were analysed by electrophoresis of blood
samples on cellulose-acetate plates (Titan III, Helena
Laboratories). The electrophoresis was run at 400 V for
20 min in citrate buffer. Stain solution was applied directly
on the plates which were incubated for about 10 min at 37°C
(Eppig et al. 1977).
Table 1. Development of diploid embryos obtained at the 2-cell stage by the fusion of the two blastomeres after the
enucleation of one of them
Experimental embryos
blastocysts (%
of 2-cell fused)
transferred to
mice that became
pregnant/
total transferred
transferred to
mice that became
pregnant/
total transferred
pregnants/
recipients
experim.
control
-
4/21
7/7
1/3
1(J16)«
7(J16)'
100%
5+2(ab)/l6
in vitro development
volts
time (s)
2-cell
fused/
total
50-85
480
24/24
pulses parameters
at least
one divis.
-
Controls
Development to term
25%
50
480
69/70
22/25
15
33/47
16/20
4/5
12/33
40/61
-
3/5
77/129
(60%)
23/27
(85%)
8/13
(62%)
ll+6(ab)/40
27-5 %
24/77
(31 %)
(68%)
40
480
92/92
26/26
185/186
(99%)
48/51
(94%)
16
36%
(62%)
TOTAL
31
(65 %)
"The mouse has been opened at 16 days of pregnancy. Experimental as well as control embryos were normal.
(ab) abortion site.
31%
-
12/23
(52%)
Diploid mouse embryos constructed from haploids
775
Separation of polypeptides by SDS-polyacrylamide
gel electrophoresis in two dimensions
Groups of 12-23 embryos were labelled by a 3 h incubation
in [35S]methionine (—SOOCimmol"1) diluted in Whitten
medium at a final concentration of SOCimmol"1. They
were rinsed three times in phosphate-buffered medium
containing 0-4 % of PVP 40 (polyvinylpyrrolidone).
Separation of proteins was carried out according to the
method of O'Farrell (1975). The ampholine composition
used in the first-dimension isoelectrofocusing was 4% of
pH3-5-10 and 1 % of pH4-6. The second-dimension
electrophoresis was carried out in an SDS gel with 12-5 %
acrylamide. The resulting gels were fixed in ethanol/acetic
acid solution and soaked for half an hour in Amplify
(Amersham) liquid for fluorography. They were dried and
exposed to Kodak XAR 5 film for 3-5 weeks.
Results
Development of embryos after electrofusion
In most of the experiments described in this paper, we
have used an electrofusion technique (see Materials
and methods). Table 1 gives the results of an experiment designed to check the efficiency of the technique and the effect of electrofusion on the survival of
2-cell embryos. One of the two blastomeres was
enucleated and the embryo was exposed for 480 /us to
an electric field of 800-1000 V cm"1 in Whitten medium and kept in this medium at 37°C. Generally,
fusion of the two blastomeres started between 5 and
15 min after the pulse and was completely achieved
after 30-60min. Fusion of the two blastomeres occurred in 99 % of the 2-cell embryos operated. 65 %
of the fused embryos reached the blastocyst stage and
31 % of the blastocysts transferred into mice that
became pregnant resulted in viable offspring compared to 52 % for nonmanipulated control embryos.
Development of diploid embryos reconstructed at the
2-cell stage from haploid parthenotes and
androgenotes
The design of the manipulations used for the reconstitution of the diploid embryos at the 2-cell stage is
described in Fig. 1.
The experiment took place between 43 and 46 h
after HCG injection, i.e. about 24h after the induction of the parthenogenetic embryos and 24-28 h
after the removal of the female pronucleus from the
fertilized eggs for the androgenote embryos. This
corresponds to the late 2-cell stage. Two-dimensional
SDS-acrylamide gels of proteins obtained from embryos labelled for 3 h with [35S]methionine at the time
of the experiment were performed to control the
activation of the genome. Haploid parthenotes and
androgenotes, as well as diploid controls, all expressed the hsp 68xlO3Mr protein, characteristic of
Fig. 1. (A) Unfertilized eggs (C57/CBA) were activated
by ethanol and the resulting haploid parthenotes with a
polar body and a pronucleus were kept in culture until
the 2-cell stage. (B) Androgenetic embryos (BALB/c/SJlx
BALB/c) were obtained by taking out the female
pronucleus of a fertilized egg. (C) One of the blastomeres
of the 2-cell haploid parthenote was enucleated. (D) A
male karyoplast from an androgenetic 2-cell embryo was
introduced underneath the zona pellucida of the
parthenote. (E) The two blastomeres together with the
male karyoplast were fused either by inactivated Sendai
virus particles or, as shown here, by electrofusion (see
Table 1). (F) After fusion, all of the embryos showing
two nuclei in the same cell were reintroduced into the
oviduct of a pseudopregnant mouse on the first day of
pregnancy. Coat colour and Gpi isozymes were used as
markers: Gpi-la: B, Gpi-lb: • , Gpi-lab: 0 .
776
J. Barra and J. -P. Renard
Fig. 2. Incorporation of
[35S]methionine into
proteins synthesized
during a 3h labelling
period by (A) 1-cell
diploid normal mouse
embryos (n=23); (B) 2-cell
diploid normal embryos
(n = 12);(C) 2-cell haploid
parthenogenotes (n = 12);
(D) 2-cell haploid
androgenotes (n=12).
Labelling was started 24 h
post-HCG in the case of
1-cell embryos (A), and
44 h post-HCG in the case
of 2-cell embryos (B-D).
Arrowheads indicate the
positions of the 68 and the
70x10*Mr proteins that
have been taken as
markers for the activation
of the embryonic genome.
Arrows show the 35K
complex. (B-D show the
results of one out of three
experiments giving
identical results.)
B
the early expression of the embryonic genome (Bensaude et al. 1983; Flachs et al. 1984), see Fig. 2.
The diploid reconstituted embryos were introduced
into one of the oviducts of pseudopregnant mice on
day 1 of gestation, while control embryos of another
phenotype were introduced into the other one (in
general, extra FjBSxBALB/c eggs were kept and
used as controls).
The results of the experiment are presented in
Table 2. Normal development of the reconstituted
embryos took place in 50% (8/16) of the mice that
became pregnant (75% (12/16) for controls). From
94 experimental embryos transferred to mice that
became pregnant, 18 (19%) developed to viable
fetuses at day 14 (n = 2) or to living offsprings
(n = 16). This percentage is lower than that observed
with nonmanipulated control embryos (39/83, i.e.
47%).
Two of the experimental newborns died during the
2 days following birth. Of the remaining 14 animals, 5
were females and 9 were males. All of them were
agouti. They all displayed the expected heterozygote
GPI-1 isozyme pattern (Fig. 3). The 14 animals have
reached adulthood and have reproduced.
Discussion
Our experiments show that male and female genomes
kept separately in haploid embryos until the late
2-cell stage are still able, when put together in the
same cytoplasmic environment, to ensure full-term
development. They also indicate that the independent activation of the two parental genomes in the
haploid embryos before the reconstruction of the
diploid ones, as evidenced by the presence of the 68K
heat-shock protein, does not prevent their necessary
coordinate expression later on.
In these experiments, the two haploid nuclei and
the cytoplasm of the parthenogenetic recipient were
Diploid mouse embryos constructed from haploids
111
Table 2. Development of diploid embryos constructed at the late 2-cell stage by the fusion of a karyoplast
containing an haploid androgenote nucleus with a 2-cell haploid parthenote a blastomere of which has been
enucleated
Experimental embryos
Controls
Fused
embryos
transferred to mice
that became pregnant/
total transferred
transferred to mice
that became pregnant/
total transferred
pregnants/
recipients
15
10
6/10
12/12
2/3
20
10
10/10
16/16
2/2
10
9
10
6/9
3/6
-
8
8/8
6/6
0/7
9/9
0/9
Late 2-cell
parthenote
recipients
7
7
9
9
17
18
17
48
0/17
0/12
12/17
37/46
8/12
10/13
1/2
0/1
2/2
2/2
0/2
2/2
0/2
0/2
0/3
2/3
3/4
179 (84%)
94/171 (55%)
83/130(64%)
16/30 (53 %)
10
14
7
11
12
9
18
20
17
51
214
Development to term
0/10
14/14
10/10
0/14
10/10
0/9
0/5
0/9
experim.
controls
l + l(ab)/6
(J14)*
l + l(ab)/10
(J15)'
l+3(ab)/l2
(J14)*
13/16
(J15)*
0/6
0/8
1/3
-
l(ab)/6
0/9
-
4/12
12/37
18/94(19%)
6/14
7/10
—
6/10
—
4/8
1/10
39/83 (47 %)
* Mice have been opened at 14 or 15 days of pregnancy. Experimental as well as control embryos were normal.
(ab) site of abortion.
b ! 2
3
4
5 6 7 8
synchronized. That the nucleus and cytoplasm be at
the same developmental stage seems to be of critical
importance for completion of the preimplantation
stage: transfer of a normal diploid nucleus from an
older blastomere to an enucleated zygote (McGrath
& Solter, 1984a; Solter et al. 1985) markedly reduced
or completely prevented the development of the
embryos. Similar results were obtained when a nucleus from a later stage was transferred to an enucleated 2-cell blastomere (Robl et al. 1985; Tsunoda et
al. 1987). However, when maternal and paternal
genomes are kept separately as haploid nuclei for
several cycles, desynchronization of one of them does
not impair development to term: a female haploid
nucleus from a 8-cell, and even in a few cases from a
16-cell stage, or a male haploid nucleus from a 4-cell
stage, transferred into an enucleated egg that had
10 11
C c
Fig. 3. Genetic origin of offsprings
was confirmed by Gpi isozyme
analysis: a, Gpi-lb: C57/CBA;
b, Gpi-la: BALB/c/SJL; 1-11,
reconstructed embryos; c, control
embryos: BALB/c/SJL x BALB/c.
kept the pronucleus of the opposite sex, permitted
full-term development of the embryos (Surani et al.
1986). Taken together these observations suggest that
very early interactions between male and female
genomes exert some commitment effect on the fate of
the resulting diploid nucleus, which becomes very
dependent on the stage of its cytoplasmic environment.
The differential contribution of the male or the
female genome is clearly observed at the cellular level
soon after implantation: parthenotes (or gynogenotes) and androgenotes display different and
complementary phenotypes. Cells containing only
the female genome participate preferentially in the
embryo proper whereas cells containing only the male
genome are essentially involved in the development
of the extraembryonic tissues (Surani et al. 1986;
778
J. Barra andJ.-P. Renard
Surani et al. 1987). It seems reasonable to postulate
from the nuclear transfer experiments summarized in
the previous paragraph that some very early interactions between the parental genomes create new
conditions for their further expression. These conditions are probably essential to ensure full-term
development. Identification of a critical stage for
maternal and paternal genome interactions is of
particular interest to pinpoint which of the molecular
differences between diploid normal embryos, parthenotes and androgenotes are determinant for normal
development.
Our present work shows that such interactions are
not required before the late 2-cell stage. By constructing diploid embryos at increasingly later cleavage
stages, we should be able to obtain evidence of a
critical step. Experiments are in progress to explore
the 4- and 8-cell stages. The use of a simple and
reliable method of electrofusion considerably increases the efficiency and even the practicability of
these experiments.
Another interesting result presented in this paper is
that the egg cytoplasm does not need any male
contribution at the 1-cell stage in order to ensure the
full development of the embryo: the recipient embryos used at the 2-cell stage are parthenotes and they
received only trace amounts of the 2-cell cytoplasm
that surrounds the male nucleus in the karyoplast. On
the other hand we cannot exclude that, in the
androgenote embryos, the male genome had interacted with some female cytoplasmic factors before its
transplantation.
In the case of the mutant strain DDK, the results of
nuclear transplantation support the notion that a
product derived from the male nucleus acts at the
pronucleus stage on the egg cytoplasm and can affect
later stages of embryonic development (Renard &
Babinet, 1986). The data reported here imply that if
such interactions are a general event they can also be
induced at the 2-cell stage.
This work was initiated in the laboratory of Professor C.
L. Markert at Yale University where J.B. was a postdoctoral fellow. She would like to thank Professor Markert for
introducing her to the world of the mouse embryo, and for
fruitful discussions. J.B. thanks the Fondation pour la
Recherche M6dicale that supported her during her stay at
Yale.
The authors are indebted to J.-L. Popot for his contribution in setting up the electrofusion conditions. They
thank C. Babinet and the members of the Unitt: de
g6n6tique des mammiferes for stimulating discussions, O.
Bensaude for advice using the 2D-gel technique and J.-P.
Changeux and P. Benoit for the loan of a GRASS S44
Stimulator.
This work was supported by a grant from the Institut
National de la Sant6 de la Recherche M6dicale (CRE n°
864002). J.-P. Renard is affiliated with the Institut National
de la Recherche Agronomique.
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{Accepted 5 January 1988)