BIOLOGY OF REPRODUCTION 49, 933-942 (1993)
Nuclear-Cytoplasmic Interactions during the First Cell Cycle of Nuclear Transfer
Reconstructed Bovine Embryos: Implications for Deoxyribonucleic
Acid Replication and Development'
K.H.S. CAMPBELL, 2 W.A. RITCHIE, and I. WILMUT
AFRC, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom
ABSTRACT
The present study investigated the decay of maturation-promoting factor (MPF) activity in electrically activated in vitromatured bovine oocytes and examined the influence of the cell cycle stage of both the donor nucleus and the recipient cytoplasm
upon the morphology and DNA synthesis potential of the donor nucleus in reconstructed embryos. The decay of MPF activity
was studied both biochemically in electrically activated in vitro-matured oocytes and by morphological examination of nuclear
structure in reconstructed bovine embryos. As measured by Hl kinase activity in groups of 10 oocytes, the level of MPF declined
rapidly to 30 + 4% (of the maximum level in unactivated control oocytes) at 60 min and reached a basal level of 20 ± 6% at
120 min. This level of activity was then maintained until at least 9 h postactivation. In contrast, when MPF activity was assayed
by morphological examination of nuclei in individual reconstructed embryos, the decline in activity occurred over a period of
9 h postactivation. DNA synthesis of nuclei arrested at the G1/S border and in G2 phases of the cell cycle was examined in
embryos reconstructed at the time of oocyte activation, i.e., when MPF levels were maximal, and by fusion 10 h postactivation,
when no MPF activity could be detected. All nuclei transferred at the time of oocyte activation underwent nuclear envelope
breakdown (NEBD) and subsequent DNA synthesis. However, when nuclei were transferred 10 h after activation, no NEBD was
observed in any nuclei. Nuclei arrested at the G1/S border or nuclei in S phase when transferred in the absence of NEBD
underwent DNA synthesis, while no DNA synthesis was observed in G2 nuclei transferred into this cytoplasmic environment.
The results presented show that all nuclei, regardless of cell cycle stage, undergo DNA replication when transplanted into
metaphase II (MeII) cytoplasts in which MPF activity is high. From these observations we would suggest that one factor that
may contribute to the present low frequency of development of bovine nuclear transfer embryos is the ploidy of the reconstructed embryo after the first cell cycle. In order to maintain correct ploidy of the reconstructed embryo, only G1 nuclei should
be transferred at the time of activation, when MPF levels are high. In contrast, when the integrity of the nuclear membrane is
maintained by transfer at 10 h postactivation, when MPF activity is absent, the rereplication of G2 nuclei is prevented and correct
ploidy of the reconstructed embryo may be maintained.
INTRODUCTION
persal of nucleoli, 4) reformation of the nuclear envelope,
and 5) nuclear swelling. Previous reports have suggested
that the induction of NEBD and PCC are essential for the
reprogramming of gene expression and that these events
increase the developmental potential of the reconstructed
embryo (e.g., in the rabbit [10]). However, the extent and
timing of each of these events vary both between species
and between nuclei at different stages of the cell cycle (e.g.,
in the rabbit [11]). Studies in the rabbit using donor blastomeres at defined cell cycle stages have shown that development to blastocyst is greater when blastomeres in G1
or early S phase are transferred than when the donor nuclei are in late S or G2 phases [10]. Additionally, morphological examination of the prematurely condensed chromatin and chromosomal constitution of the reconstructed
embryos suggests that when G1 or early S phase nuclei are
transferred there is little effect on the chromosomal constitution of the reconstructed embryo. However, when late
S phase nuclei are transferred, a large proportion of blastomeres in reconstructed embryos contain both large chromosomes and other abnormalities [11]. A possible explanation for these observations can be obtained from
previously reported studies on cell cycle regulation with
particular reference to the cytoplasmic control of nuclear
events and of cellular DNA replication by maturation-pro-
During the last decade, nuclear transfer in mammals has
become an important tool both for study of fundamental
aspects of development (e.g., [1, 2]) and for embryo multiplication (e.g., [3]). The technique of nuclear transfer involves the fusion of a single nucleated blastomere or cell
(karyoplast) to a recipient cytoplast prepared by enucleation of a metaphase II (MeII) oocyte or a one-cell zygote.
In ungulate species, the use of MeII oocytes as cytoplasts
has become the method of choice (reviewed in [4, 5]). In
these species, fusion of the cytoplast and karyoplast is induced by the application of an electric pulse that also induces activation of the enucleated oocyte.
The morphological events that occur in the donor nucleus after transfer into an enucleated MeII oocyte have been
studied in a number of species including mouse [6], rabbit
[7], pig [8], and cow [9]. In summary, these include 1) induction of nuclear envelope breakdown (NEBD) followed
by 2) premature chromosome condensation (PCC), 3) disAccepted June 23, 1993.
Received April 5, 1993.
'This project was supported by a consortium made up of Animal Biotechnology,
Cambridge; Milk Marketing Board, Meat and Livestock Commission and the Department of Trade and Industry.
2
Correspondence. FAX: (031) 440 0434.
933
934
CAMPBELL ET AL.
moting factor (MPF). MPF was first described as an activity,
present in the metaphase cytoplasm of maturing oocytes
and unfertilized eggs of frogs, that could induce maturation
of immature oocytes when microinjected [12]. When heterokaryons were produced by fusion of vertebrate cells
synchronized at different points during the cell cycle, the
subsequent cell cycle was either delayed or advanced depending on the cell cycle phases of the cells fused and the
relative volumes of their cytoplasm. Mitotic cells induced
NEBD and PCC when fused with cells in G1, S, or G2 phases,
suggesting that mitotic cells also contained an activity that
could determine the behavior of nuclei from interphase cells
[13, 14]. Further studies have shown that the onset of M phase
(mitosis/meiosis) is regulated by a mechanism common to
all eucaryotic cells (for review see [15]). MPF has been
identified as a complex of two proteins, cyclins and p34d 2;
p3 4cdc2 is a protein kinase whose kinase activity is regulated
by changes in its phosphorylation state and by its association with cyclins. Throughout the cell division cycle, the
level of p3 4 cdc2 remains constant, but the level of cyclins
varies. In vivo, activation of p34 cd2 kinase triggers entry of
the cell into M phase and results in NEBD, chromosome
condensation, reorganization of the cytoskeleton, and
changes in cell morphology. In vitro, p34d 2 has been shown
to phosphorylate a number of proteins including histone
H1, nuclear lamins, RNA polymerase II, p60 sr, T antigen,
and elongation factor. The in vitro phosphorylation of histone H1 has been used as the basis of a biochemical assay
for the estimation of p34 ~d 2 activity (for reviews see [15,16]).
During a single cell cycle, all genomic DNA must be replicated once and only once prior to mitosis. If any of the
DNA either fails to replicate or is replicated more than once,
then the ploidy of that nucleus at the time of mitosis will
be incorrect. The mechanisms by which replication is restricted to a single round during each cell cycle are unclear; however, several lines of evidence imply that maintenance of an intact nuclear membrane is crucial to this
control. In studies of heterokaryons, analysis of interphase
fusions indicated that S phase cells advanced G1 nuclei into
S phase but that G2 nuclei could not be induced to re-enter
S [17,18]. In more recent studies, the replication capacity
of G1 and G2 nuclei has been examined through use of a
cell-free DNA replication-competent extract from Xenopus
eggs [19, 20]. In this system, intact G1 nuclei replicate their
DNA semiconservatively, while intact G2 nuclei do not replicate. However, if the nuclear membranes of the G2 nuclei
are permeabilized by treatment with detergent, then they
also replicate; this suggests that the integrity of the nuclear
membrane is required to distinguish G2 from G1 nuclei.
In heterokaryons when NEBD and PCC are induced in S
phase nuclei by fusion to mitotic cells, DNA synthesis continues on the condensed chromatin [17]; similarly in the
Xenopus cell-free system when NEBD and PCC are induced
in partially replicated nuclei, then DNA synthesis is also seen
to continue [21].
Mature mammalian oocytes are arrested in metaphase of
the second meiotic division and contain high levels of MPF
activity (i.e., mouse [22]). Upon fertilization or activation,
the level of MPF activity declines; this loss of activity precedes the decondensation of the male and female chromatin and the formation of pronuclei. Previous studies have
shown that immediately upon fusion to a MeII oocyte, NEBD
and PCC occur in the donor nucleus (i.e., cattle [9]). Thus
we can hypothesize that this removal of the nuclear membrane will remove the block to rereplication of previously
replicated DNA and will lead to the occurrence of DNA synthesis in donor nuclei from all phases of the cell cycle, including those from S or G2. However, if nuclear transfer
were performed after the decline of MPF activity, then the
integrity of the nuclear membrane would be preserved, and
regulated replication of all nuclei should occur.
In the present study we have examined the decline of
MPF activity in activated in vitro-matured bovine oocytes
(using in vitro phosphorylation of histone Hi) and also in
nuclear transfer embryos reconstructed at different times
following activation (by use of morphological criteria). We
have then assessed the replication of nuclei arrested at the
G1/S border or in G2 phase of the cell cycle, transplanted
in the presence and absence of MPF activity.
MATERIALS AND METHODS
Oocyte Maturation and Fertilization
Ovaries were obtained from a local abattoir and maintained at 28-32 0C during transport to the laboratory. Cumulus oocyte complexes (COC) were aspirated from follicles 3-10 mm in diameter via a hypodermic needle (1.2mm internal diameter) and placed into sterile plastic universal containers. The universal containers were placed into
a warmed chamber (35°C) and the follicular material was
allowed to settle for 10-15 min before three-quarters of the
supernatant was poured off. The remaining follicular material was diluted with an equal volume of dissection medium (TCM 199 with Earle's salts [Gibco, Grand Island, NY],
75.0 mg/L kanamycin, 30.0 mM Hepes [pH 7.4, osmolarity
280 mOsmol/kg H20]) supplemented with 10% bovine
serum, then transferred into an 85-mm petri dish and examined for COCs under a dissecting microscope. Complexes with at least 2-3 compact layers of cumulus cells
were selected, washed three times in dissection medium,
and transferred into maturation medium (TCM 199 with
Earle's salts [Gibco], 75 mg/L kanamycin, 30.0 mM Hepes,
7.69 mM NaHCO 3 [pH 7.8, osmolarity 280 mOsmol/kg H2 01)
supplemented with 10% bovine serum and 1 x 106 granulosa cells/ml. COC were cultured for 24-30 h on a rocking table at 39 0C in an atmosphere of 5% CO 2 in air.
At 24 h after the onset of maturation, fertilization was
carried out according to the method described by Vergos
et al. [23]. Briefly, oocytes were gently pipetted in order to
remove adhering granulosa cells and to break up aggre-
DNA SYNTHESIS IN NUCLEAR TRANSFER BOVINE EMBRYOS
gated COC. Disaggregated COC were then washed once and
transferred into 50-R1 microdrops of fertilization medium
(5-15 oocytes/drop) containing sperm (1.5 x 10 6 /ml).
Embryo Culture and Synchronization
At 36-40 h after coincubation of the spermatozoa and
oocytes, embryos were sorted on the basis of developmental stage by use of a dissecting microscope. Embryos
were selected for synchronization into G1/S or G2 cell cycle
stages or transferred to Hepes-buffered M2 medium [24] at
room temperature for storage until used for nuclear transfer. For synchronization of 8-cell blastomeres at the G1/S
border, 4-cell embryos were cultured in 5 ptg/ml Nocodazole (Sigma Chemical Co., St. Louis, MO) in bovine oviduct epithelial cell (BOEC)-conditioned medium [25] for
15 h; embryos that remained at the 4-cell stage were then
released into fresh medium, without Nocodazole, containing 5 mM hydroxyurea (Sigma) and monitored hourly for
cleavage. Cleaved embryos were transferred to M2 at room
temperature for storage until use. For synchronization of
8-cell blastomeres in the G2 phase of the cell cycle, 8-cell
embryos were cultured in the presence of 5 g/ml cycloheximide for 12 h; embryos remaining at the 8-cell stage
were then transferred to M2 at room temperature for storage until used for nuclear transfer.
ElectricalActivation of In Vitro-Matured Bovine Oocytes
Matured oocytes were stripped of cumulus cells by gentle
pipetting in hyaluronidase (400 U/ml; Sigma) in dissection
medium, washed three times in dissection medium, washed
once in activation medium (0.3 M mannitol, 0.1 mM MgSO 4,
0.05 mM CaCl2 ), transferred into an activation chamber, and
given a single pulse of 1.25 kV/cm for 80 Rjsec. After activation, oocytes were washed once in dissection medium
and cultured under light paraffin oil (BDH) in microdrops
(25 lI) of BOEC-conditioned medium at 39°C in 5% CO2
in air.
935
tion chamber, which was at room temperature. A donor
blastomere was inserted under the zona pellucida of an
enucleated oocyte; the couplet was then transferred to the
activation chamber as described above. The couplet was
manually aligned in the chamber and fusion was induced
by a single electrical pulse of 1.25 kV/cm for 80 Rsec. Couplets were then returned to culture medium and monitored for fusion.
MorphologicalExamination
Oocytes, activated oocytes, and nuclear transfer embryos
were wet mounted on ethanol-cleaned glass slides under
coverslips, which were attached by use of a mixture of 5%
petroleum jelly and 95% wax. Mounted embryos were then
fixed for 24 h in freshly prepared methanol:glacial acetic
acid (3:1), stained with 45% aceto-orcein, and examined by
phase-contrast and DIC microscopy via a Nikon MicrophotSA
Assessment of MPFActivity by Histone HI Kinase Assay
Groups of 10 oocytes were placed into 10 Rpl of histone
kinase buffer (80 mM -glycerophosphate, 20 mM EGTA,
15 mM MgCI 2, 1 mM dithiothreitol), immediately snap frozen in a methanol CO 2 bath, and stored at -20°C until assayed. For H1 kinase assay, samples were thawed on ice;
10 1l of assay solution (0.6 mM ATP, 2.0 mg/ml histone
H1, 1.85 MBq 32P-ATP [110 TBq/mmol; Amersham, Arlington Heights, IL]) was added, and the tube contents were
mixed well by pipetting and then incubated at 37°C for 15
min. After incubation, the reaction was stopped by placing
the samples on ice. Samples were spotted onto small pieces
of P81 paper (Whatman, Clifton, NJ), washed three times
for 20 min each in 150 mM phosphoric acid, washed twice
in industrial methylated spirit (IMS), and then air dried. Incorporation of 3 2P-phosphate was determined by placing the
dried filters in 3 ml of scintillation fluid (Optiphase Hisafe
[LKB, Rockville, MD]) and counting in a Wallac 1410 scintillation counter.
Enucleation and Nuclear Transfer
Oocytes to be used as recipients for nuclear transfer were
enucleated as previously described [26, 27]. Briefly, oocytes
at 24-28 h of maturation were stripped of cumulus cells
and cultured in medium containing cytochalasin B (7.5 ,ag/
ml; Sigma) and Hoescht 33342 (5.0 ,ig/ml; Sigma) for 15
min at 39 0C in an atmosphere of 5% CO 2 in air. Enucleation
was carried out in the same medium, without Hoescht, at
room temperature by aspirating a small amount of cytoplasm from directly beneath the first polar body. To confirm enucleation, the aspirated cytoplasm was checked by
epifluorescence for the presence of the meiotic spindle and
polar body. Enucleated oocytes were then returned to maturation medium at 39°C until 30 h post-onset of maturation
(hpm). For nuclear transfer, oocytes were removed from
the maturation medium and transferred to the manipula-
Assessment of MPF Activity by Nuclear Transfer
Enucleated oocytes were activated as described above
and fused to blastomeres from asynchronous 8-cell embryos at the time of activation and at various times following activation. Fused couplets were incubated for 60 min
and then mounted, fixed, stained, and examined for nuclear morphology as described above.
Determinationof DNA Synthesis
Embryos were incubated in BOEC-conditioned medium
containing 100 pzM 5-bromo-2'-deoxyuridine 5'-triphosphate (BrDU; Sigma) for varying periods of time. After incubation, the zona was removed from the labeled embryos
by incubation in 0.5% protease in dissection medium. Zonafree embryos were washed in PBS, transferred onto cover-
936
CAMPBELL ET AL.
100
I
80 -
7
a
t
0
i
60 -
E
0
o
aR
40 -
Y
X
20
20n-
20
·
30
·
·
40
·
·
)0
50
Time
Maturation
Time
(hours)
FIG. 1. Effect of time in culture on the number of in vitro-matured bovine oocytes that are arrested at metaphase of the second meiotic division
(open symbols) and activation response (solid symbols). The graph represents the mean ( SEM) of three batches of oocytes (> 20 oocytes per
group per batch).
slips, and allowed to air dry. Dried embryos were then fixed
with methanol at -20°C for 20 min, washed in PBS, permeabilized in 0.1% Triton X-100 for 2 min at room temperature, washed in PBS, hydrolyzed in 4 N HC1 for 30 min
at room temperature, and again washed in PBS. Excess PBS
was removed and the coverslips were placed into a humidified chamber. Embryos were then incubated in the
humidified chamber with 50 pl of primary antibody (rat
anti-bromodeoxyuridine, Seralabs, Crawley, U.K.), diluted
1:10 in PBS containing 1% fetal calf serum (FCS), overnight
at 4°C. Coverslips were then washed three times in PBS and
incubated with the secondary antibody (FITC-conjugated
rabbit anti-rat [Sigma]) diluted 1:50 in PBS containing 1%
FCS for 4 h at room temperature. After incubation, the
coverslips were washed three times in PBS, excess PBS was
removed, and the coverslips were mounted on glass slides
with DABCO (Sigma).
Post
Activation (Mins)
FIG. 2. Decline of histone H1 kinase activity in electrically activated in
vitro-matured bovine oocytes. The graph represents the mean activity (-+
SEM) of three batches of oocytes (10 oocytes per batch per time point).
Oocytes were treated and assayed as described in the text; the graph shows
the results of assays conducted in the presence (open symbols) or absence
(closed symbols) of cAMP-dependent protein kinase inhibitor.
Disappearanceof MPF Activity
The rate of disappearance of MPF activity as measured
by H1 kinase activity and the rate of pronuclear formation
were compared in three batches of in vitro-matured oocytes. In all three batches, Hi kinase activity rapidly declined immediately after activation, to approximately 30 +
4% of the maximum level at 60 min postactivation and to
20
6% at 120 min postactivation (Fig. 2). This decline in
activity preceded the onset of the formation of pronuclei
(Fig. 3). Significant variation between batches of oocytes
was observed in the rate of MPF decline and also in both
the rate and the timing of pronuclear formation. The decline of MPF activity was also assessed by morphological
criteria in individual reconstructed bovine embryos (see Fig.
4). The results from these experiments are presented in
Figures 5 and 6. As can be seen, there is considerable variation in the induction of NEBD both between and within
batches of oocytes. As stated above, the object of these ex-
RESULTS
Oocyte Maturation and Activation
In preliminary experiments, groups of bovine oocytes
matured for differing periods of time were examined by
aceto-orcein staining for meiotic maturation and the ability
to be activated by a single electrical pulse of 1.25 kV/cm.
The results from these experiments are summarized in Figure 1. At 24 h after onset of maturation, the majority of
oocytes are at MeII; however, it is not until 30 h that a significant percentage of these oocytes became activated as
demonstrated by the formation of a single pronucleus. In
addition, on prolonged incubation the number of oocytes
that were maintained at MeII decreased. In subsequent experiments, oocytes were matured for 30 h.
E
0
i
E
U.
0
U
100
Time
20uu
post
3u
4UU
activation
5uu
ouu
(mina)
FIG, 3. Pronuclear formation in in vitro-matured electrically activated
bovine oocytes. Oocytes from the same batches as in Figure 2 were fixed,
stained, and examined for pronuclear formation.
DNA SYNTHESIS IN NUCLEAR TRANSFER BOVINE EMBRYOS
937
have artificially arrested the nuclei of 8-cell blastomeres at
the G1/S interface; 4-cell embryos arrested at mitotic metaphase by treatment with Nocodazole (5 pzg/ml) were released into medium containing 5 mM hydroxyurea. After
cleavage, the decondensation of chromatin and the reformation of a nuclear membrane were confirmed microscopically in groups of fixed embryos, and the absence of
DNA replication was confirmed by bromodeoxyuridine incorporation (data not shown).
G2-arrested 8-cell blastomeres were prepared by incubation of in vitro-produced 8-cell embryos in BOEC-conditioned medium containing 5 g/ml cycloheximide. When
asynchronous 8-cell embryos were treated, DNA synthesis
was observed for up to 12 h post-treatment. In contrast,
when treated embryos were incubated in bromodeoxyuridine after 12-20 h of cycloheximide treatment, no incorporation was observed. These results suggest that cycloheximide treatment has no adverse effects on the DNA
replication and that the nuclei arrest in a post-S premitotic
state. In a group of embryos incubated in cycloheximide
for 15 h and then released into fresh medium containing
bromodeoxyuridine, 80% (12 of 15) of the embryos continued division. DNA replication was observed in 31% (15
of 48) of divided blastomeres within 2 h of release and in
71% (32 of 45) of divided blastomeres by 4 h after release.:
In order to minimize exposure to cycloheximide, ,embryos
were monitored for cleavage and transferred to medium.
containing cycloheximide 10 h after cleavage. Incubation
was then continued in the presence of cycloheximide for
12 h. Arrested embryos were then transferred to Hepesbuffered M2 medium containing 10% FCS until used as nuclear donors.
FIG. 4. DIC microscopy of aceto-orcein-stained bovine embryos reconstructed at the time of activation and after the disappearance of MPF activity. Figure 4a shows NEBD and PCC in a nucleus transplanted at the time
of activation. Figure 4b shows an intact nucleus transplanted after the disappearance of MPF. Reconstructed embryos were fixed and stained as described in Materials and Methods.
periments was to assess the replication potential of G1 and
G2 nuclei by determining a period after activation at the
end of which no NEBD was observed in any of the reconstructed embryos; from the data presented in Figures 3 and
4, a period of 10 h postactivation was chosen.
Synchronization of Donor Embryos at the G1/S Border
and in G2 Cell Cycle Phases
During the early cleavage stages of bovine embryos, the
G1 period of the cell cycle is either absent or extremely
short [2], and at any one time within an unsynchronized
population of embryos, most nuclei will be in S phase. We
DNA Synthesis in Reconstructed Bovine Embryos
An experiment was performed in order to examine the
replication potential of nuclei at different phases of the cell
cycle transplanted into different cytoplasmic environments
during the first cell cycle of in vitro-matured bovine oocytes. Eight-cell, in-vitro-produced bovine embryos were
synchronized at the G1/S border or in G2 phases of the
cell cycle. Such synchronized blastomeres were 1) fused to
enucleated unactivated in vitro-matured bovine oocytes at
30 h after the onset of maturation and 2) fused to enucleated oocytes 10 h after the activation of these oocytes
(i.e., 40 h after onset of maturation). Fused couplets were
incubated in BOEC-conditioned medium containing 100 VM
BrDU for 12 h (10-22 h postactivation).
When nuclei arrested at the G/S border or in the G2
phase of the cell cycle were transferred to either an unactivated oocyte or an activated oocyte in which it was anticipated that NEBD would not occur, the occurrence of DNA
synthesis was influenced by the cell cycle phase of both the
recipient cytoplasm and the donor nucleus. DNA synthesis
was observed when nuclei at the G1/S phase border were
transferred into either cytoplasmic environment (21 of 21
938
CAMPBELL ET AL.
_
-
EXPT1
*·-
EXPT2
_
10
8
EXPT3
6
c"
z
4
*-
EXPT4
a---
EXPT5
2
100
0
Post
Time
200
300
Activation
(mins)
400
FIG. 5. Nuclear envelope breakdown (NEBD) in reconstructed bovine embryos. In vitro-matured bovine oocytes
were enucleated as described in the text. At 30 h post-onset of maturation, oocytes were activated with a single
electrical pulse of 1.25 kV/cm for 80 tLsec. Activated oocytes were fused to blastomeres from unsynchronized 8-cell
in vitro-produced bovine embryos at various times after activation by the application of a second electrical pulse (1.25
kV/cm, 80 ipsec). Fused couplets were cultured for 60 min, fixed, stained, and examined for NEBD as described in
the text. Results show the percentage of embryos undergoing NEBD from five separate batches of in vitro-matured
bovine oocytes.
unactivated, 16 of 17 activated). When G2 nuclei were transferred, DNA synthesis was observed when unactivated bovine oocytes were used as recipient cytoplasts (14 of 14);
but no DNA synthesis was observed when activated oocytes
were used as recipients (0 of 25). When unsynchronized 8cell embryos were used as nuclear donors, bromodeoxyuridine incorporation was observed in 100% of transfers at
0 h (18 of 18) and in 100% of transfers at 0 h + 10 h (19
of 19), suggesting that these nuclei were in G1 or S phase
at the time of transfer and that at 10 h after transfer in the
absence of NEBD, DNA replication continued.
One criticism of these experiments is that the absence
of bromodeoxyuridine incorporation observed when G2
am
z
nuclei were transferred after the disappearance of MPF activity is that the recipient cytoplast is unable to replicate any
nucleus. Therefore, experiments were also carried out using unenucleated activated oocytes as recipients for nuclear
transfer. This experimental protocol allows the replication
of the female pronucleus to be used as a positive control
for the replication potential of the oocyte cytoplasm. As observed when enucleated oocytes were used as donors, all
nuclei, regardless of cell cycle stage, incorporated bromodeoxyuridine when transferred into unactivated oocytes
(G1/S, 17 of 17; G2, 21 of 21) as shown by the presence
of two BrDU-labeled nuclei (Fig. 7a). However, when pronuclear eggs were used as recipients, nuclei arrested at the
G1/S border replicated their DNA (18 of 19) but G2 nuclei
did not rereplicate (2 of 23); thus only one nucleus became
BrDU labeled (Fig. 7b). The presence of intact nuclei was
checked in all BrDU-positive and BrDU-negative embryos
by DIC microscopy (Fig. 7, c and d). Thus in a single cytoplasmic environment we have shown that replication potential is controlled by a nuclear factor.
at
DISCUSSION
0
1 00
Time
200
3 00
4 00
Post Activation
5 00
600
(Mins)
FIG. 6. Decline of MPF activity as assessed by NEBD in reconstructed
bovine em bryos. Results (mean + SEM) include those for the batches of
5 plus .subsequent analyses.
in Figure
oocytes rei presented
.
,
In the present study we have shown that when bovine
~~
embryos are reconstructed by nuclear transfer into lell
oocytes at the time of activation, NEBD occurs in all donor
nuclei, and that this is subsequently followed by reformation of the nuclear membrane and DNA synthesis. In contrast, when nuclear transfer is performed into previously
enucleated and activated oocytes, i.e., after the decline of
,~~
!i'
,
....-T
DNA SYNTHESIS IN NUCLEAR TRANSFER BOVINE EMBRYOS
939
FIG. 7. Assessment of DNA replication in reconstructed bovine embryos and confirmation of the presence of an intact nucleus. a) BrDU incorporation
in the transplanted nucleus and the female pronucleus of an unenucleated reconstructed bovine embryo fused at the time of activation (x300). b) Replication
in a single nucleus in a bovine embryo reconstructed 10 h postactivation using a G2 nucleus (x150). c and d) confirmation by DIC microscopy of the
presence of an intact nucleus (d; x600) in the absence of BrDU incorporation (c; x600).
940
CAMPBELL ET AL.
ACTIVAT ION
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-
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DNA SYNTHESIS IN NUCLEAR TRANSFER BOVINE EMBRYOS
MPF activity, then the integrity of the nuclear membrane is
maintained. G1 nuclei replicate their DNA, and S phase nuclei continue DNA replication; however, rereplication of G2
nuclei does not occur (these results are summarized in Fig.
8). These results suggest one possible contributory factor
to the present low frequency of development reported following nuclear transfer into MeII oocytes, i.e., that nuclei
at any phase of the cell cycle other than G1 at the time of
transfer will rereplicate previously replicated DNA and aneuploidy will thus result. This explanation is supported by
previous reports showing that the frequency of development to blastocyst of reconstructed rabbit embryos is greater
when donor nuclei are in the G1 phase of the cell cycle
than when they are in S phase [9].
The objective of nuclear transfer in ungulate species is
to produce genetically identical offspring. In general, in vivoproduced embryos at the 32-64-cell stage are used as nuclear donors. In such embryos, only a small proportion of
cells are expected to be in the G1 phase of the cycle. However, the experiments reported here raise the possibility of
producing reconstructed embryos of normal ploidy from
donor nuclei that are at any point in the cell cycle by performing nuclear transfer into enucleated activated oocytes
in which MPF activity has declined. This situation is analogous to the use of 1-cell mouse zygotes as recipients for
nuclear transfer. With fertilized 1-cell mouse zygotes, in
contrast to ungulate embryos, it is possible to easily visualize and remove the pronuclei. In such zygotes, MPF activity has declined as shown by the presence of pronuclei.
Previous studies have shown identical rates of development
when karyoplasts from early and late 1-cell zygotes are
transplanted into early and late 1-cell cytoplasts, respectively. In contrast, when karyoplasts were prepared from 2cell embryos, the frequency of development was greater
when cytoplasts late in the first cycle were used as recipients, regardless of the donor cell cycle stage [28].
In the present study we have shown that by maintaining
the integrity of the nuclear membrane, DNA synthesis is
inhibited when G2 nuclei are transferred whereas G1 and
FIG. 8. Schematic representation of events during the first cell cycle
of electrically activated bovine oocytes and the potential effects of cell cycle
stage of both the donor karyoplast and the recipient cytoplast upon DNA
replication in the donor nucleus. Bovine oocytes are arrested at metaphase
of the second meiotic division and contain high levels of MPF activity. Upon
activation, MPF activity declines, the second polar body is extruded, the
chromatin decondenses, and a pronucleus is formed. The pronucleus subsequently undergoes DNA replication. When embryos are reconstructed at
the time of activation, the levels of MPF activity are high; all nuclei undergo
NEBD and PCC. These events are followed by chromatin decondensation
and reformation of the nuclear envelope. The diagram shows the potential
replication pattern and DNA content of nuclei from defined cell cycle stages.
When transferred at the time of activation (group A), only nuclei that are
in G1 at the time of transfer would appear to maintain the correct DNA
content at the end of the first cell cycle. In contrast, when nuclei are transferred after the decline of MPF activity, no NEBD is observed and coordinated replication of nuclear DNA in nuclei from all cell cycle stages should
occur (group B). 2C = diploid, 4C = tetraploid, 8C = octoploid.
941
S phase nuclei continue synthesis. In these experiments we
have not examined the ploidy of reconstructed embryos,
nor are we able to distinguish whether small amounts of
synthesis are occurring in the G2 nuclei. However, we would
suggest that by maintaining the integrity of the nuclear
membrane, the correct ploidy of the reconstructed embryos should be maintained regardless of the cell cycle stage
at the time of transfer. To increase the frequency of successful nuclear transfer when MeII oocytes are being used
as cytoplasts, a population of G1 donor nuclei would be
required. At the present time, techniques for the synchronization of embryos are unreliable; however, the isolation
of embryonic stem (ES) cell lines would provide a large
number of cells that could possibly be synchronized and
used as nuclear donors.
Previous studies have suggested that exposure of the donor chromatin to the oocyte cytoplasmic environment is
required for nuclear reprogramming. In Xenopus it has been
shown that an increased frequency of development is obtained when nuclei are transferred into oocytes at first
meiotic metaphase (reviewed in [29]). The possibility that
exposure to the oocyte cytoplasmic environment is required for nuclear reprogramming cannot be ruled out. (AUS:
Deleted two sentences that were identical to two in previous paragraph; okay?) To further increase the exposure
of the donor chromatin to the host cytoplasmic environment, nuclear transfer into oocytes at earlier stages of maturation or into MeII oocytes in the absence of activation
may be required. After leaving the reconstructed oocyte for
a period of time it would be necessary to induce activation.
These opportunities to prolong the period of exposure to
oocyte cytoplasm may enhance the developmental potential
of the reconstructed embryo.
The variability in the decline of MPF activity in activated
oocytes, both between and within batches, and the differences between activated oocytes and reconstructed embryos observed in the present studies suggest that other
factors associated with either the maturation or the activation of oocytes in vitro may contribute significantly to the
present low frequency of embryo development. The MPF
decline that was observed in reconstructed embryos occurred over a period of 9 h; in contrast, Hi kinase activity
in activated oocytes had declined to basal levels at 2 h postactivation. The slower disappearance of MPF activity observed in reconstructed embryos may be interpreted in a
number of ways. Firstly, interactions between cytoplast and
karyoplast cytoplasm may increase or decrease the decay
of MPF directly; this may depend on the cell cycle phase
of the karyoplast. Secondly, the increase in cytoplasmic volume of the reconstructed embryo may alter MPF decay. An
alternative hypothesis is that the decay of MPF activity differs in enucleated oocytes as a result of either enucleation,
the manipulations involved in enucleation, or an alteration
in the activation potential of the enucleated oocyte. Further
investigations of activation in oocytes, enucleated oocytes,
942
CAMPBELL ET AL.
and reconstructed embryos may help to separate these effects. Additionally, the development of activation protocols
that closely mimic the oocyte responses induced by sperm
penetration may help to increase the success of nuclear
transfer in ungulate species.
In summary, these experiments establish that by combining karyoplasts with cytoplasts at appropriate phases of
the cell cycle it is possible to ensure normal ploidy of reconstituted embryos: cells at any stage of the cell cycle may
be fused with cytoplasts after disappearance of MPF. Thus
when nuclear transfer is carried out using unsynchronized
32-64-cell embryos as nuclear donors, the use of an enucleated activated oocyte may provide a "universal cytoplast"
that can minimize the consequences of cell cycle effects.
Further studies are needed to determine the developmental potential of embryos produced in these ways.
ACKNOWLEDGMENTS
We are grateful to Owen Ravie, Karen Mycock, and Patricia Ferrier for skilled
technical assistance.
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