Laboratory of Nuclear Reprogramming and Cell Differentiation, Department of Obstetrics and Gynaecology, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074, Singapore
Author for correspondence (e-mail:
scng{at}embryonics.biz)
Accepted 11 February 2004
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SUMMARY |
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Key words: Somatic cell nuclear transfer, Cell cycle, Spindle formation, Non-human primate, Embryo transfer
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Introduction |
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Although successful production of animal clones from somatic cells has been
achieved in many species, including sheep
(Wilmut et al., 1997), cattle
(Cibelli et al., 1998
), mice
(Wakayama et al., 1998
), goat
(Baguisi et al., 1999
), pig
(Onishi et al., 2000), cat (Shin et al.,
2002b
), rabbit (Chesne et al.,
2002
), mule (Woods et al.,
2003
) horse (Galli et al.,
2003
) and rat (Zhou et al.,
2003
), there has been no success in non-human primates. In
addition, although blastocysts have been developed from rhesus embryos after
SCNT (Mitalipov et al., 2002
),
only nuclear transfer of embryonic cells in non-human primates has resulted in
live-births (Meng et al.,
1997
).
The success of SCNT depends on several parameters that impact on the
ability of the cytoplast to reprogram the nucleus of the donor cell, or to
reverse the epigenetic changes that occur during development
(Wilmut et al., 2002).
Quiescent G0 donor cells were used during initial SCNT experiments
(Wilmut et al., 1997
).
However, SCNT has also been achieved with donor cells in G1
(Cibelli et al., 1998
), G2
(Wakayama et al., 1999
) and
G2/M (Ono et al., 2001
). In
mice (Wakayama et al., 1998
;
Gao et al., 2002
), bovine
(Shin et al., 2002a
) and
rabbit (Chesne et al., 2002
),
it had been reported that there was spindle formation after somatic donor cell
introduction into the enucleated oocyte, and that misaligned metaphase plates
were also observed. By contrast, it was reported that primates were different
from other animals, as disarrayed abnormal mitotic spindles with misaligned
chromosomes were formed in all SCNT embryos, and no pregnancies resulted from
SCNT embryos transferred into surrogates
(Simerly et al., 2003
). It was
suggested that meiotic spindle removal may be the source of primate SCNT
anomalies, and primate NT appears to be challenged by stricter molecular
requirements [Nuclear Mitotic Apparatus (NuMA) and Kinesin-related protein
HSET (human spleen, embryo, testes)] for mitotic spindle assembly than does NT
in other mammals (Simerly et al.,
2003
). Although cell cycle co-ordination in cloned embryos by NT
was reviewed previously (Campbell et al.,
1996
), there has been no data about spindle and nuclear formation
published in non-human primates, and there are also no reports on the events
in the first cell cycle of SCNT embryos.
In this study, we describe the first cell cycle changes of SCNT embryos in non-human primates for the first time. Our data demonstrate that SCNT embryos of the non-human primate are similar to other animals in that they can form a normal PCC spindle. We also report early pregnancy failures after embryo transfers of such reconstructed embryos.
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Materials and methods |
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Establishment and culture of donor cell
Tissue sources
Skin biopsy specimens were derived from a 180-day-old male M.
fascicularis fetus and an adult male Lion-tailed Macaque (M.
silenus). Fresh cumulus cells were obtained from the follicles of the
macaques that had oocyte recoveries (ORs).
Establishment and culture of fibroblast cells
Skin biopsy specimens were washed in Ca2+- and
Mg2+-free Dulbecco PBS (Invitrogen) and minced into pieces. Tissue
pieces were cultured in DMEM (Invitrogen) supplemented with penicillin,
streptomycin and 10% (v/v) FBS (Invitrogen), and cultured at 37°C in 5%
CO2. Tissue pieces were removed using a 30G needle when cells with
a fibroblast-like morphology started to migrate out of the tissues. After
reaching 100% confluency, cell monolayers were disaggregated using PBS
containing 0.15% (w/v) trypsin and 1.8 mM EDTA, then were passaged twice more
before being frozen in DMEM with 20% FBS and 10% (w/v) DMSO (Sigma), and
stored in liquid nitrogen.
Fibroblast treatments and flow cytometric analysis of the cell cycle
Cell culture flasks (75 cm3) were plated with frozen/thawed
fibroblasts at 1-3x106 cells/flask. At 70-80% confluency,
cells were fixed in ethanol. Other cells were grown to 100% confluency and
then treated as follows before being fixed: (1) serum starved by culturing in
DMEM + 0.5% FBS for another 2 or 5 days; or (2) cultured in regular growth
medium that was changed every 2-3 days for another 2 or 5 days to promote
contact inhibition. For fixation, cells from each treatment were
disaggregated, pelleted by centrifugation, resuspended and then slowly mixed
with 4.5 ml of cold 70% (v/v) ethanol. After 12 hours of ethanol fixation at
4°C, cells were stained in PBS containing 0.1% (v/v) Triton X-100, 0.2
mg/ml of RNase A and 20 mg/ml of propidium iodide (Sigma), for 15 minutes at
37°C. Cells were analyzed using an Epics-Elite flow-analyzer (Coulter,
USA). Cell cycle phases were calculated using Winmdi version 2.8, based on the
PMT4 histogram.
Ovarian stimulation and macaque oocytes recovery
Procedures for superovulation of the Long-tailed Macaque (LoTM, M.
fascicularis) and collection of their oocytes have been described
previously (Ng et al., 2002).
The female monkeys were hyperstimulated with a fixed regimen comprised
initially of downregulation with a GnRH agonist, triptorelin (Decapeptyl,
Ferring, Kiel, Germany). A dose of 1.8 mg active triptorelin per 2 kg body
weight was administered intramuscularly. Two weeks later, human recombinant
follicle stimulating hormone (rFSH; Gonal-F, 75 IU, Serono, Geneva) was
administered at 37.5 IU per 2 kg body weight daily for 12 days. On the last
day of FSH treatment, 1000 IU human chorionic gonadotropin (hCG; Profasi,
Serono, Geneva) was administered intramuscularly to each monkey. Oocyte
recovery was then performed 34-36 hours after hCG treatment, During the OR
procedure monkeys were sedated with Zoletil 100 (Virbac, Peakhurst, Australia)
at 8 mg per kg body weight. Laparoscopic recovery of the oocytes was performed
with a 4 mm Storz laparoscope attached to a video system. The oocytes were
aspirated with a double-lumen needle (FAS Set C2, Gynetics Medical Products
NV, Hamont-Achel, Belgium) attached to the Cook aspiration and flushing
systems (VMAR 5100 and V-MAR 4000, respectively; Cook Australia, 12
Electronics Street, Brisbane Technology Park, Eight Mile Plains, Queensland
4113, Australia). The cumulus-oocyte complexes (COCs) were collected in
HEPES-buffered Ferticult Flushing Medium (FFM), washed, and then cultured in
Ferticult IVF medium (FertiPro NV, 8730 Beemem, Belgium) in 5% CO2
in air. Oocytes stripped of cumulus cells by exposure to hyaluronidase (80
IU/ml; Type IV-S bovine testes, Sigma-Aldrich, St Louis, MO, USA) were placed
in medium IVF-20 (Vitrolife), at 37°C in 5% CO2 until further
use.
SCNT procedures
Enucleation
Recipient MII oocytes were loaded individually into 5 µl droplets of
HEPES-buffered IVF medium (Ferticult, Belgium) containing 10 µg/ml
cytochalasin B (Sigma). A small opening on the zona pellucida was made by
acidic Tyrode solution (pH 1.8) and then the second meiotic spindle was
aspirated with a small volume cytoplasm (<1% of oocyte) under polarized
microscopy (SpindleView, CRI, MA). After enucleation, the karyoplast was
stained with 1 µg/ml Hoechst 33342. Maternal chromosome removal was
confirmed twice by DNA epifluorescence imaging.
Nuclear transfer
LoTM fresh cumulus and starved fetal skin fibroblast cells, as well as
starved LiTM adult skin fibroblast cells, were used as donor cells for nuclear
transfer. I.D. spiked pipettes, 8 µm and 5 µm, were used for fibroblast
and cumulus cells, respectively. Single donor cells, collected from the 10%
PVP droplet, were ruptured by gentle aspiration out of the injection needle,
and then directly microinjected into the enucleated oocyte.
Activation
Cells were induced 2 hours after microinjection by electric pulses, and
then after a further 2 hours combined with 5 µM ionomycin (Sigma) or 7%
ethanol for 5 minutes. Two consecutive direct current pulses (1.5 kV/cm, 50
µseconds) were delivered by a BTX Cell Manipulator 2001 (Genentronics, San
Diego, CA).
SCNT embryo culture
All SCNT embryos after manipulation were cultured in IVF-20 (Vitrolife) at
37°C with 5% CO2, 5% O2 and 90% N2. After
activation, SCNT embryos were cultured in IVF-20 containing 5 µM
cytochalasin B and 10 µg/ml Cycloheximide for 5 hours, then in IVF-20.
14-16 hours after activation, nucleus formation was checked before transfer to
G1.2 (Vitrolife). 24 hours later, all SCNT embryos were transferred to G2.2
(Vitrolife). After culture for another 28-30 hours, 4- to 8-cell SCNT embryos
were selected for replacement.
Embryo transfer and pregnancy monitoring
The procedure for embryo transfer of the reconstructed embryos has been
described previously (Ng et al.,
2002). Three days after SCNT, selected cleaved embryos were
replaced into the fallopian tube of the monkey from whom oocytes were
recovered earlier. Laparoscopic tubal embryo transfer (TET) was performed
using a homemade embryo transfer catheter, a flexible polythene tube (o.d.,
1.09 mm; i.d., 0.38 mm), threaded through a 25 g hypodermic needle. Luteal
phase support was provided by 10 mg progesterone administered intramuscularly
for 14 days starting on the day of OR. Pregnancies were ascertained by fetal
ultrasound, with the presence of a viable gestational sac and heart beat.
Imaging
Periodically, reconstructed oocytes were stained immunocytochemically to
observe cytoskeletal organization and DNA configuration
(Shin et al., 2002a). Controls
included non-immune and secondary antibodies alone. ß-Tubulin antibody
was used as a primary antibody to detect microtubules. Laser-scanning confocal
microscopy was performed using a Zeiss LSM500, equipped with Argon and
Helium-Neon lasers for the simultaneous excitation of FITC-conjugated
secondary antibodies (Sigma) and propidium iodide DNA stain.
Statistical analysis
Results were analyzed using the Pearson's 2 test.
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Results |
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Discussion |
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Understanding the first cell cycle changes is crucial in designing
strategies for successful SCNT in different species. The coordination of
nuclear and cytoplasmic events during embryo reconstruction was reviewed
previously (Campbell et al.,
1996). Our data suggests the following sequence: the donor cell
nuclear membrane breaks down because of the direct and indirect effects of MPF
upon the transferred nucleus; the chromatin in G1/G0 condenses into a
premature condensed chromosomal state; and then microtubule assembly occurs to
form a PCC spindle from oocyte signals that would have formed the second
meiotic spindle if the maternal chromosomes were still present. It also
confirms that somatic cell DNA in non-human primates can form a normal PCC
spindle after the somatic cell is introduced into a enucleated oocyte, as is
seen in mice (Gao et al.,
2002
) and bovine (Shin et al.,
2002a
).
In this study, 70.4% of transferred somatic cells underwent chromosome
condensation following their introduction into an enucleated MII oocyte,
microtubule assembly occurred in 68.5% of reconstructed embryos, and 14.8% of
them had normal PCC spindles with 2 poles. PCC spindle formation is influenced
both by the donor cell and by the oocyte. Poor quality oocytes may not be able
to initiate nuclear membrane breakdown of the somatic cell, chromosome
condensation or microtubule assembly. Our data is suggestive of problems in
both chromosome condensation and microtubule assembly in SCNT embryos.
Misaligned metaphase plates were found in 9.3% of reconstructed embryos in
which microtubules were normally assembled with 2 poles, but the prematurely
condensed chromosomes were improperly captured. In mice
(Wakayama et al., 1998), and
rabbit (Chesne et al., 2002
),
such misaligned metaphase plates were also observed. This may due to lack of,
or inadequate levels of, certain factors, such as HSET
(Simerly et al., 2003
).
The age of the oocytes could be a very important factor that affects results. Aging oocytes may not be able to reprogram the somatic cell. The majority of oocytes used in this study were collected at 34-37 hours after hCG administration, and were then enucleated 2 hours later. 21% (143/684) of oocytes used for enucleation had just reached the MII stage (extruded the first polar body) before enucleation. In some oocytes that were enucleated 4-5 hours later, unusual changes were observed, including fusion of 2-cell embryos back to 1-cell embryos with two nuclei. This suggests that cytoskeleton changes in aging oocytes may affect cell dynamics after reconstruction.
Nuclear formation rate in this study was only 36.5%, although chromosome
condensation and microtubule assembly occurred in 70% of reconstructed
embryos. This suggests that nuclear reformation is a problem in a large number
of such embryos. Nuclear formation in fertilization following nuclear transfer
is controlled by the oocyte cytoplasm
(Collas, 1998). However, the
sequence of events leading to nuclear reformation in cloned embryos is
unclear, as are the factors influencing nuclear reformation. We have observed
a one-day delay in nuclear formation in this study. Interestingly, there were
some embryos without nuclear formation after cell injection that cleaved or
became fragmented. It is possible that there was no chromosome duplication,
which thus resulted in fragmentation; another possibility is that the S phase
was too short and that they were missed. Further studies are needed.
The chromosome and cell cycle changes postulated on the basis of our data
are for the injection technique. These changes may be different when using the
cell fusion method (electro-fusion), as the direct current used for fusion may
partially activate the oocyte. Although cell injection may provide a
mechanical stimulus, a gentle technique will minimize that. Reprogramming
factors, as yet unknown, in the MII oocyte are capable of remodeling the fully
differentiated somatic nucleus, although time is needed. Hence, activation 2
hours after the introduction of the somatic nucleus may be essential for donor
cell reprogramming (Wilmut et al.,
2002).
To date, there has been no successful live birth from SCNT in non-human
primates. This lack of success was recently postulated to be due to the
removal of molecular signals during enucleation, especially of NuMA and HSET
(Simerly et al., 2003).
Primate NT appears to be challenged by stricter molecular requirements for
mitotic spindle assembly than are needed in other mammals. Our data suggest
that this may not necessarily be the case.
NuMA (Nuclear Mitotic Apparatus) is an intranuclear matrix protein (Zeng et
al., 2000) that has kinase-recognition motifs, including sites for
cAMP-dependent kinase, PKC, CDC2 (MPF) and Ca2+/calmodulin kinase,
in the C-terminal domain of the protein
(Yang and Snyder, 1992) and
that is present in the nucleus during interphase
(Compton et al., 1992
). During
mitosis, NuMA is essential for the terminal phases of chromosome separation
and/or nuclear reassembly (Compton et al.,
1992
; Price et al., 1986). It is abundant in the cell
(Compton et al., 1992
), and
hence in donor cells too. After the cell is introduced into an enucleated
oocyte, NuMA from the donor cell should assist in forming a normal spindle.
Simerly et al. reported that, in the non-human primate, all reconstructed
SCNTs and ECNTs examined displayed aberrant spindles, and that NuMA was not
detected in abnormal spindles (Simerly et
al., 2003
). We believe this may be due in part to technical
problems, such as excessive aspiration of the cytoplasm (we remove less than
2% of the cytoplasm with the Spindle View System). Species difference may
partially explain the difference, and culture environment may also be a
contributing factor, as an optimal medium for SCNT has not been reported. In
fact, our data supports the conventional belief that incomplete nuclear
re-programming is likely to be the reason for the lack of live births in
primates.
From our experiments, successful implantation implies the ability to
develop embryonic stem cells and undergo early gastrulation. However, there
are many possible reasons for failure to develop to term in SCNT, as have been
reviewed previously (Wilmut et al.,
2002). Developmental manipulations (including SCNT) or
non-physiological culture environments may result in inappropriate epigenetic
modification of imprinted genes during early embryogenesis when many
allele-specific imprints were established or maintained
(Young and Fairburn, 2000
),
including defective fetal reprogramming in livestock
(Young et al., 2001
). Data has
been conflicting, reflecting the state of uncertainty regarding gene
expression following SCNT. In SCNT bovine embryos, normally expressed
metabolic enzyme genes have been reported
(Winger et al., 2000
), as well
as the abnormal expression of genes essential for early embryonic development
(Daniels et al., 2000
). Such
abnormal expression has also been reported in cloned mouse blastocysts or ES
cells (Boiani et al., 2002
),
and genes important to early development frequently fail to be activated in
mouse embryos cloned from adult cells
(Bortvin et al., 2003
),
suggesting that aberrant transcription patterns detected in cloned embryos may
lead to abnormalities at various embryonic stages.
As it is likely that there are many differences among species, we need to
optimize the protocol for primates. The success of ECNT
(Meng et al., 1997) has
confirmed that enucleation is not a problem for NT. Meng et al. used embryos
generated by in vitro fertilization as donor cells, cryo-preserved the
reconstructed embryos and then transferred them into synchronized surrogates;
they obtained two live births from 29 reconstituted embryos. However, it is
still possible that spindle-associated factors may play a role, and, if so, a
minimal enucleation procedure will be crucial. Thus, new strategies are
needed. An approach whereby the donor cell is introduced before the oocyte DNA
is removed (`reverse-order' cloning method) has been described by Peura et
al., and results in significantly higher blastocyst rates in the sheep (Peura
et al., 2003). However, Wakayama recently reported that this reverse-order
cloning method had no effect on cloned-embryo development in mouse
(Wakayama et al., 2003
); these
findings suggest that neither oocyte chromosome depletion per se, nor the
potential removal of `reprogramming' factors during enucleation, explain the
low efficiency of NT cloning.
In conclusion, there is a crucial need to understand the molecular and cellular events that occur after the introduction of a somatic cell into an enucleated oocyte. It is only with this understanding that it will be possible to use this technique to its maximal potential.
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ACKNOWLEDGMENTS |
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Footnotes |
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