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JEZ 788
250
Y.THE
TSUNODA
JOURNAL
ANDOF
Y. KATO
EXPERIMENTAL ZOOLOGY 278:250–254 (1997)
Full-Term Development After Transfer of Nuclei
From 4-Cell and Compacted Morula Stage Embryos
to Enucleated Oocytes in the Mouse
Y. TSUNODA* AND Y. KATO
Laboratory of Animal Reproduction. College of Agriculture and Research
Institute for Animal Developmental Biotechnology. Kinki University, Nara,
631, Japan
ABSTRACT
The developmental ability of enucleated mouse oocytes reconstituted between the
nucleus from 4-cell mouse embryos at different cell stages and recipient cytoplasms at different
conditions, and the developmental ability of oocytes receiving nuclei from compacted morulae were
examined. The highest development was observed with the nucleus at the G1 stage fused with
cytoplasm at the M stage. Although the enucleated oocytes receiving a nucleus from 4-cell embryos did not develop after transfer to a recipient female, young were obtained after renuclear
transfer to enucleated fertilized eggs. Live mice were also obtained from the nucleus of compacted
morula. J. Exp. Zool. 278:250–254, 1997. © 1997 Wiley-Liss, Inc.
Since the first report of a nuclear transfer sheep
by Willadsen (’86), nuclear transfer has been done
in farm animals by fusing enucleated oocytes at
the second metaphase with a blastomere of donor
preimplantation embryos. The developmental ability of nuclear transferred eggs is influenced by
the cell cycle of the donor nucleus and the donation of the recipient cytoplasm. The nuclei fused
with recently ovulated or mature oocytes show
premature chromosome condensation (PCC) with
dispersal of individual duplicate chromosomes, reform pronucleus-like nucleus after parthenogenetic activation, duplicate chromosomal DNA and
division to the 2-cell stage (Tsunoda et al, ’89;
Collas et al., ’92; Campbell et al., ’93). Only the
G 1 nuclei are suitable for maintaining correct
policy after nuclear transfer (Campbell et al., ’93).
Nuclei fused with preactivated oocytes did not
show PCC, but in the nuclei at the G1 stage, not
G2 stage, DNA replicated, and in the nuclei at the
S stage DNA duplication continued (Campbell et
al., ’93; Barnes et al., ’93; Takano et al., ’96). In
this case, a nucleus at any cell stage can be used
as the donor nucleus. The developmental ability
of nuclear transfer to activated oocytes was higher
than that after transfer to nonactivated oocytes
(Kono et al., ’94). After serial nuclear transfer in
cattle, more than 40 blastocysts were produced
from one cattle embryo (Takano et al., unpublished
observation) and identical calves were produced
(Bondioli et al., ’90).
In contrast to the success in cattle, the success© 1997 WILEY-LISS, INC.
ful production of offspring by nuclear transfer is
limited in the mouse. Cheong et al. (’93) obtained
young after transfer of enucleated oocytes fused
with nuclei from 2-, 4-, and 8-cell stage embryos.
They found that the developmental ability was
higher in enucleated oocytes receiving nuclei at
an early stage of the cell cycle than at the middle
or late stage. However, the effect of the condition
of recipient cytoplasm in connection with the
stage of cell cycle of donor nucleus on the development of reconstituted oocytes has not been
reported. Although a nucleus of a blastomere
of morula (Rossant and Vijh, ’80) and the inner
cell mass cell of blastocysts (Gardner, ’68; Kato
and Tsunoda, ’95) can produce chimeric mice
after aggregation with precompacted embryos
or after injection to blastocysts, live mice have
not been obtained after nuclear transfer beyond
the 8-cell stage.
In the present study we examined: (1) the developmental ability of oocytes reconstituted between the nucleus from 4-cell mouse embryos at
different cell stages and recipient cytoplasms at
different conditions, and (2) the developmental
ability of oocytes receiving nuclei from compacted
morulae.
*Correspondence to: Dr. Y. Tsunoda, Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara, 631, Japan.
Received 23 October 1996; Revision accepted 8 January 1997
NUCLEAR TRANSFER OF MOUSE EMBRYOS
MATERIALS AND METHODS
Donor embryos
Two-cell stage embryos were obtained from superovulated F 1(C57BL/6 × CBA) female mice
mated with F1 males 45 hours after injection of
human chorionic gonadotrophin (hCG). The embryos were stored at 4°C for 7 to 8 hours in M2
(Fulton and Whittingham, ’78) supplemented with
10% fetal calf serum (FCS). Then they were cultured in M16 medium (Whittingham, ’71) supplemented with nocodazole (3 µg/ml) for 14 to 17
hours at 37°C in 5% CO2 and 95% air atmosphere
(Kato and Tsunoda, ’92). The treated embryos
were washed several times with M2 and M16 and
cultured in M16 for 1.5 hours. Some of them were
incubated with 5 µg/ml aphidicolin for 1 hour to
suppress the DNA synthesis (Spindle et al., ’85).
Most 2-cell embryos divided into the 4-cell stage
during the treatment. The nuclei of such embryos
should be considered at the G1 stage. The other
2-cell embryos were culture for 1.5 to 2.0 hours
(0.5 to 1.0 hours after cleavage, G1 to S stage), 3.5
to 4.0 hours (early S stage), 6.5 hours (middle S
stage) and 12.5 hours (late S to G2 stage) in M16
before nuclear transfer. The nucleus with a small
volume of cytoplasm (karyoplast) from 4-cell embryos was used for nuclear transfer.
Morula stage embryos were obtained after a 24hour culture in M16 of 8-cell embryos, recovered
from F1 females mated with F 1 males, 65 hours
after hCG injection. They were treated with M16
supplemented with nocodazole for 6 hours and
their zonae pellucidae were removed with 0.5%
pronase. Zona-free embryos were pipetted into
single blastomeres in trypsin-EDTA solution. After
further culture for 30 minutes, divided blastomeres
were immediately used for nuclear transfer. To inhibit DNA synthesis in a divided blastomere,
aphidicolin was added in some cases to the media
for all procedures from pronase treatment to single
blastomere culture.
Recipient oocytes
Recipient oocytes were collected from superovulated F1 females 16 to 19 hours after the injection of hCG, and denuded by the treatment with
hyaluronidase (300µ/ml) in M2 and washed three
times with M2. Observation of oocytes under an
inverted microscope with Nomarski optics revealed a small swelling around the metaphase
chromosomes. The zonae pellucidae around the
swelling was cut by using Narishige micromanipulators (Narishige, Japan) and the chromosomes
251
were removed as previously reported (Tsunoda et
al., ’86; Tsunoda and Kato, ’95). The enucleated
oocytes were immediately used for recipient oocytes, or after parthenogenetic activation. For activation, enucleated oocytes were transferred to
Zimmerman’s cell fusion medium (Wolfe and
Kraemer, ’92) in a chamber comprised of two wire
electrodes mounted 1 mm apart on a glass slide
(BTX, U.S.A.). Two DC pulses of 50 V/mm for 50
µseconds at 2-second intervals were given three
times at 20-minute intervals. After electrical
stimulation, the oocytes were cultured in M16 for
2.5 to 3.5, 5.5 to 6.5 or 10 to 11 hours before
nuclear transfer.
In some experiments, oocytes with chromosomes
were activated, cultured in M16 containing cytochalasin B (CB, 5 µg/ml) for 6.5 hours and used
as the recipient cytoplasm at the pronuclear stage.
The fertilized eggs in vitro (Tsunoda and Chang,
’75) were also used at a pronuclear stage 6.5 to
10 hours after insemination. The pronuclei of activated or fertilized eggs were removed before
nuclear transfer.
Nuclear transfer
A single karyoplast from a 4-cell embryo or blastomere from compact morulae was fused with an
enucleated oocyte by using inactivated Sendai virus (HVJ; Tsunoda et al., ’86). The unfused
karyoplast or blastomere pairs were further induced to fuse by electrical stimulation with DC
pulses of 50V/mm for 50 µseconds. All oocytes used
before activation were electrically stimulated three
times at the same conditions for preactivation.
When the nucleus at the G1 stage of the cell cycle
was used, aphidicolin was added to all media used
until fusion. The total aphidicolin treatment period was less than 3 hours. In a preliminary experiment, we observed that this treatment had
no effect on the developmental ability of 2-cell
mouse embryos in vitro.
Some eggs that developed to the morula or blastocyst stage were transferred to the oviducts of
day 1 pseudopregnant CD-1 strain females and
killed on day 18.5 to examine the fetuses.
Renuclear transfer into enucleated
zygotes or 2-cell embryos
When the reconstituted oocytes developed to
pronuclear stage, the pronucleus was fused again
with in vitro fertilized (F1 females and males)
enucleated cytoplasm by HVJ. Some reconstituted
oocytes forming a pronucleus were cultured to 2cell stage. In half of the oocytes, one nucleus of
252
Y. TSUNODA AND Y. KATO
each reconstituted eggs at 2-cell stage was fused
with the enucleated blastomere of an in vitro fertilized 2-cell stage embryo by HJV and the enucleated remaining blastomere of the 2-cell embryo
was left without a nucleus. In the remaining oocytes, both nuclei of the reconstituted eggs at 2-cell
stage were fused with each enucleated blastomere
of fertilized 2-cell embryos. The 2-cell embryos
with fused blastomeres were discarded. The
renuclear transferred eggs were cultured for 3 or
4 days in vitro and some eggs developed to morula
or blastocyst stage were transferred to the oviducts of day 1 pregnant CD-1 strain females
mated with the same strain males, which were
subsequently allowed to go to term.
stage was used as a donor. Most of the oocytes
that had been activated 10 to 11 hours before
nuclear transfer became fragmented shortly after nuclear transfer. Forty-three morulae and
blastocysts obtained in various groups were
transferred to eight recipient mice but none of
them developed to young.
To inhibit the fragmentation after nuclear
transfer, a parthenogenetic or fertilized egg at
the pronuclear stage was fused with a nucleus
at various cell stages shortly after removal of
the pronucleus. However, none of them developed to morula or blastocyst stages (0/119 for
activated and 0/54 for fertilized eggs, data not
shown in Table 1).
RESULTS
Renuclear transfer of nucleus
from 4-cell embryos
The effect of the combination of cell cycle stage
of donor nucleus and cytoplasm on the development of the enucleated oocyte receiving a nucleus
from a 4-cell embryo was examined.
As shown in Table 1, the developmental ability of the nuclear transferred embryo was influenced by the combination of the cell cycle
stage of the donor nucleus and condition of cytoplasm of enucleated oocyte. The proportion of
the nonactivated oocytes (0 hour) receiving the
G 1 nucleus (0 hour) developed to morula and
blastocyst stage was significantly (P<0.05) by
χ2 test) higher than those obtained in the other
groups (23% vs. 0% to 10%). In the oocytes
nonactivated or activated 2.5 to 3.5 hours before nuclear transfer, the developmental ability after nuclear transfer was low regardless of
the cell cycle of the donor nucleus, expect the
G 1 stage. In the oocytes activated 5.5 to 6.5 and
10 to 11 hours before nuclear transfer, 2 to 10%
developed to morula and blastocyst stages, respectively, except when the nucleus at the G2/S
As shown in Table 2, 25% to 62% and 14% to
43% of renuclear transferred embryos developed
to morula and blastocyst stages, respectively.
After transfer to recipient, 8% to 36% of the
embryos developed to young in all groups except one. Live young were obtained even from
the donor embryos without aphidicolin treatment to suppress DNA synthesis after cleavage
to 4-cell stage.
Nuclear transfer of compacted morulae
The proportion of oocytes developed to 2-cell
stage after nuclear transfer without aphidicolin
treatment (74/199, 37%) was significantly (P<0.001)
lower than that obtained with aphidicolin treatment (92/162, 57%). As shown in Table 3, 18% to
26% and 5% to 18% developed to morula and blastocyst stages, respectively. After transfer to recipients, two normal live young (one male and one
female) were obtained.
TABLE 1. Developmental ability of enucleated oocyte receiving a nucleus from a 4-cell embryo in vitro
Cytoplasm
(h after activation)
0
2.5~3.5
5.5~6.5
10~11
1
Nucleus (h after division)1
0
71/88
20/88
37/40
0/40
57/68
7/68
9/66
1/66
1.5~2.0
(81)
(23)
(93)
(0)
(84)
(10)
(14)
(2)
12/32
0/32
14/54
2/54
52/79
7/79
6/49
3/49
(38)
(0)
(26)
(4)
(66)
(9)
(12)
(6)
3.5~4.0
4/29
0/29
2/44
0/44
23/59
1/59
12/49
4/49
(14)
(0)
(5)
(0)
(39)
(2)
(24)
(8)
6.5
12.5
4/42 (10)
0/42 (0)
0/41 (0)
0/41 (0)
20/61 (33)
4/61 (7)
9/56 (16)
4/56 (7)
0/21 (0)
0/21 (0)
1/24 (4)
0/24 (0)
0/32 (0)
0/32 (0)
0/27 (0)
0/27 (0)
Upper figures indicate no. of 2-cell embryos/fused (%) and lower figures indicate no. of morulae and blastocytes/fused (%)
NUCLEAR TRANSFER OF MOUSE EMBRYOS
253
TABLE 2. Developmental ability of renuclear transferred embryo receiving a nucleus from a 4-cell embryo in vivo
With or
without
aphidicolin
Stage of
renuclear
transfer
No. of eggs
fused
–
1-cell
2-cell half1
2-cell both2
1-cell
2-cell half1
2-cell both2
20
33
21
57
28
33
+
1
2
Developed to (%)
morula
10
13
13
25
7
18
blastocyst
(50)
(39)
(62)
(44)
(25)
(55)
3
8
9
14
4
11
No. of
recipients
(15)
(24)
(43)
(25)
(14)
(33)
6
5
7
17
2
5
No. of live
young/
transferred (%)
1/6
1/13
4/11
4/25
0/6
2/15
(17)
(8)
(36)
(16)
(0)
(13)
A nucleus from a reconstituted 2-cell egg was fused with a blastomere of enucleated 2-cell embryos.
Two nuclei from reconstituted 2-cell eggs were fused with both blastomeres of enucleated 2-cell embryos.
DISCUSSION
to the nuclear membrane since parthenogenetic or
fertilized zygotes, whose pronuclei were removed
immediately before nuclear transfer, fused with a
nucleus at the S stage, did not develop.
Effect of the combination of cell cycle
between donor nucleus and
recipient cytoplasm
For the normal development of nuclear transferred embryos, the nuclear information must
change from the advanced stage to fertilization.
For the recipient cytoplasm, unfertilized oocytes
at the second metaphase stage have been mainly
used (Campbell et al., ’96). In the present study,
the highest development of nuclear transferred
oocytes was obtained when the nucleus at the G1
stage was fused with the cytoplasm at the M stage
as suggested by Cheong et al. (’93). The nuclear
membrane break down (NMBD) and premature
chromosome condensation (PCC) of donor nucleus
occurred soon after fusion. However, 2% to 10%
of oocytes at the S stage which received a nucleus
at any stage except late S/G2 developed to morulae
or blastocysts. In these cases, neither NMBD nor
PCC were observed. Since the developmental process of nuclear transferred embryos was not different, the process of NMBD and PCC is not necessary
for the conversion of nucleus to the condition at fertilization. A cytoplasmic reprogramming factor(s)
besides maturation promoting factor (MPF) is possibly present in the cytoplasm of unfertilized oocytes. Such a factor(s) might be located at or bind
Full-term development of nuclear
transferred oocytes receiving a nucleus from
4-cell and compacted morula stage embryos
Cheong et al., (’93) reported that 22% of enucleated oocytes at the M stage fused with a nucleus
from early 4-cell embryos developed to young. The
present study was not in agreement with their
report. The most likely explanation for the discrepancy is the difference in the method used to
fuse the donor blastomere with cytoplasm of recipient oocytes, HVJ in the present study and electrical stimulation in the report of Cheong et al.
(’93). When the pronuclei or nucleus(ei) originating from the 4-cell embryos were fused again with
enucleated cytoplasm of fertilized zygotes or 2-cell
embryos, 8% to 36% of the morulae and blastocysts developed to young. Tsunoda and Shioda
(’88) reported that the developmental potential of
reconstituted parthenogenetic eggs which received
pronuclei of fertilized eggs was lower than that of
reconstituted fertilized eggs. Unknown factors
that enhance the developmental ability of reconstituted eggs might be present in the cytoplasm
of fertilized eggs.
TABLE 3. Developmental ability of renuclear transferred embryos receiving a nucleus from compacted morulae in vivo
With or
without
aphidicolin
Type of1
renuclear
transfer
–
both
half
both
half
+
1
Developed to (%)
No. of eggs
fused
morula
38
22
46
22
7 (18)
4 (18)
12 (26)
4 (18)
blastocyst
2
1
8
4
(5)
(5)
(17)
(18)
A nucleus from a reconstituted 2-cell egg was fused with a balstomere of enucleated 2-cell embryos.
No. of
recipients
6
1
5
2
No. of live
young/
transferred (%)
0/9
0/2
2/8
0/2
(0)
(0)
(25)
(0)
254
Y. TSUNODA AND Y. KATO
Although the sample size was small, the fact
that live young were obtained after renuclear
transfer of compacted morulae indicates that developmental totipotency of nucleus on the mouse
germ line is maintained at least up to the morula
stage. The cells used in the present study are considered to be at the blastocyst stage since individual cells separated at the compacted morula
stage were used immediately after the next cell
division.
ACKNOWLEDGMENTS
We thank Dr. K. Tanaka, Institute for Molecular and Cellular Biology, Osaka University, for providing HVJ. The present study was supported by
a grant from the Japanese Ministry of Agriculture, Forestry and Fisheries (Biotechnology and
Reproductive Technology), Special Coordination
Funds for Promoting Science and Technology from
the Science and Technology Agency, a Grant-inAid for Scientific Research No. 0856046 and the
Human Science Foundation.
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