1. Journal of Reproduction and Development, Vol. 54, No. 5, 2008, 20049
—Full Paper—
Development of Interspecies Cloned Monkey Embryos Reconstructed
with Bovine Enucleated Oocytes
Chanchao LORTHONGPANICH1)
, Chuti LAOWTAMMATHRON1)
,
Anthony Wing Sang CHAN2)
, Mariena KETUDAT-CAIRNS1)
and Rangsun PARNPAI1)
1)
Embryo Technology and Stem Cell Research Center, Suranaree University of Technology, Nakhon Ratchasima 30000,
Thailand and 2)
Division of Neurosciences, Yerkes National Primate Research Center, Emory University, School of Medicine,
Atlanta, GA 30329, USA
Abstract. This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes
and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey
embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine
enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then
cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and
monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages
using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and
bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the
16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from
the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage.
These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could
not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the
culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in
cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.
Key words: Bovine oocyte, Embryo, Interspecies cloning, Monkey, Oct-4 transcription factor
(J. Reprod. Dev. 54: 306–313, 2008)
stablishment of embryonic stem cells (ESCs) from nuclear
transferred (NT) non-human primate (NHP) embryos has yet
to be perfected [1]. Because the availability of NHPs is limited, the
cost of NHP research can be prohibitive. Therefore, an alternative
source of recipient cytoplasm that can support the reprogramming
of NHP nuclei would effectively remove this constraint and
deserves further investigation. Several studies have shown that the
ooplasm of the bovine, rabbit, sheep, domestic cat and ovine can
support early development of embryos produced by NT using
somatic cell nuclei derived from different mammalian species [2–
16]. Recent successes in live offspring born from clones of gaur [6,
9], mouflon [10] and African wildcat [14] have demonstrated that
interspecies somatic cell nuclear transfer (iSCNT) can be used to
preserve endangered species. Among the different choices of
oocytes, the bovine oocyte is one of the most popular recipient
cytoplasts for iSCNT because a large number of oocytes can be
retrieved from ovaries which can be easily obtained from an abat-
toir. Most important, the in vitro culture system for bovine
embryos is well established. Several research reports have demon-
strated that the bovine oocyte is a good candidate for use as the
recipient cytoplast for iSCNT from donor cells of sheep, pigs, rats
and monkeys [4], buffaloes [13], yaks [15] and Antarctic minke
whales [16], which all support iSCNT embryo development. An
optimal in vitro culture media is one of the most important keys to
the success of iSCNT because supplementation of appropriate
nutrients, energy sources and growth factors is critical for the
development of iSCNT embryos. The need for species-specific
embryo culture media is a widely accepted concept that has already
been demonstrated in different species; Example of this include use
of North Carolina State University-23 (NCSU-23) medium for pig
embryos [17, 18] Chatot, Ziomek, Bavister medium (CZB) [19],
potassium simplex optimized medium (KSOM) [20–22] and modi-
fied synthetic oviduct fluid with amino acids (mSOFaa) [23, 24] for
mouse embryo; Charles Rosenkrans 1 (CR1) [25] for bovine
embryos; and Connaught Medical Research Laboratories (CMRL)
[26] and hamster embryo culture medium-9 (HECM-9) [27, 28] for
NHP embryos.
In those cloned zygotes, the cytoplasmic mRNA of up to an
eight-cell stage embryo under the appropriate culture conditions is
derived from the recipient cytoplasm instead of the donor nucleus.
Cloned embryos begin to synthesize mRNA based on the genetic
materials of the donor nucleus at the eight-cell stage [29, 30].
Hence, in the case of an iSCNT embryo, an appropriate culture
media that accommodates dynamic changes in metabolic needs
during early preimplantation should be able to enhance develop-
mental competence. Additionally, Nichols et al. (1998) reported
that embryos deficient in Oct-4 are unable to develop normally and
that ICM formation at the blastocyst stage is also affected [31].
Until recently, only a limited number of studies on the expression
pattern of Oct-4 in bovine embryo have been reported [32–37].
Therefore, our goals were to determine if somatic cell nuclei of
Accepted for publication: May 19, 2008
Published online in J-STAGE: July 1, 2008
Correspondence: R. Parnpai (e-mail: rangsun@g.sut.ac.th)

2. 307MONKEY iSCNT DEVELOPMENT
monkeys could be reprogrammed after they were reconstructed
with bovine enucleated oocytes and to compare the effects of
HECM-9 (monkey embryo culture media) and mSOFaa (bovine
embryo culture media) on monkey iSCNT embryos. The Oct-4
distribution in monkey iSCNT embryos was also investigated in
this study.
Materials and Methods
Chemicals and media
All reagents were purchased from Sigma-Aldrich Chemical (St.
Louis, MO, USA), unless otherwise specified. The Emcare holding
medium (ICP Bio, Auckland, New Zealand) was used as the
manipulation medium for all manipulations at normal atmosphere
including preparation of 7% ethanol for activation. Zimmermann’s
fusion medium was used for electrofusion. HECM-9 and mSOFaa
media were used for culture of embryos.
Monkey iSCNT using a bovine enucleated oocyte as the
recipient cytoplast
Donor cell preparation: Skin fibroblasts from a crab-eating
monkey (Maccaca fascicularis) were used in this experiment.
Biopsies were kept in a modified Dulbeccco’s phosphate buffer
saline (mDPBS) at 4–8 C during transport to the laboratory. Skin
tissues were cut into small pieces (about 1 mm2
) before being
placed in a 60-mm culture dish (Nunc, Wiesbaden, Germany) and
covered with a glass slide. Five milliliters of alpha-Minimal
Eagle’s medium (α-MEM) with 10% fetal bovine serum (FBS;
Gibco BRL, Grand Island, NY, USA) was added to the dishes,
which were then cultured under a humidified atmosphere of 5%
CO2 in air at 37 C for 8 to 10 days. Skin fibroblast outgrowth was
dissociated using 0.25% trypsin/EDTA in Ca2+
- and Mg2+
-free PBS
and seeded in a 25-cm2
culture flask (Nunc). At subconfluence, the
fibroblasts were harvested by standard trypsinization, and culture
was continued in a 25-cm2
culture flask. Skin fibroblasts were fro-
zen in 10% dimethyl sulfoxide (DMSO) in normal culture media at
the third passage and stored in liquid nitrogen. Frozen-thawed
fibroblasts were cultured in a 25-cm2
culture flask and used as
nuclear donor cells up to the eighth passage. A few minutes before
injection, the actively dividing donor cells were harvested and
resuspended in Emcare holding medium. Bovine skin fibroblasts
were used as a control and were prepared by the same method.
Recipient cytoplast preparation: Bovine ovaries obtained from a
local abattoir were kept in 0.9% NaCl during transport to the labo-
ratory. The oocytes were aspirated from 2–8 mm diameter
follicles. Cumulus oocyte complexes (COCs) were matured in an
in vitro maturation medium for 22 h as described by Parnpai et al.
[38]. The cumulus cells were mechanically removed by repeat
pipetting using a fine-tip pipette in 0.2% hyaluronidase. The
oocytes were then washed five times in Emcare holding medium
before enucleation.
Somatic cell nuclear transfer: The in vitro matured bovine
oocytes were placed in Emcare medium containing 5 μg/ml cytoch-
alasin B (CB) for 15 min. The zona pellucida above the first polar
body was cut with a glass needle. A small volume (about 5 to 10%)
of cytoplasm underneath the first polar body was squeezed out.
Complete enucleation was confirmed by staining the squeezed out
cytoplasm with 5 μg/ml Hoechst 33342. Individual monkey or
bovine donor cells (diameter: 14 to 16 μm) were inserted into the
perivitelline spaces (PVS) of bovine enucleated oocytes, and fusion
was achieved by placing the couplets in Zimmerman’s fusion
medium [39] followed by electrical stimulation using two DC
pulses at 24 V for 15 μs using an SUT F-1 (Suranaree University of
Technology) fusion machine. The reconstructed embryos were
activated with 7% ethanol for 5 min and cultured in mSOFaa sup-
plemented with 3 mg/ml bovine serum albumin (BSA), 1.25 μg/ml
cytochalasin D (CD) and 10 μg/ml cycloheximide (CHX) at 38.5 C
in 5% CO2 in air for 5 h.
Parthenogenetic activation (PA)
At 26 h after in vitro maturation, the matured bovine oocytes
were activated with 7% ethanol for 5 min, and then culture was
continued in mSOFaa supplemented with 3 mg/ml BSA, 1.25 μg/
ml CD and 10 μg/ml CHX at 38.5 C in 5% CO2 in air for 5 h.
In vitro embryo culture
Monkey and bovine donor cells successfully fused to the bovine
enucleated oocytes were named monkey iSCNTs and bovine
SCNTs, respectively. Reconstructed monkey iSCNTs and bovine
SCNTs were then cultured in HECM-9 and mSOFaa at 37 and 38.5
C, respectively. Since the maternal embryonic transition in bovine
occurs at the eight-cell stage, sequential media specific to the
bovine and monkey were also used accordingly. The six culture
media and conditions designated for the monkey iSCNT embryos
are described below.
Treatment 1:HECM-9 under 5% O2, 5% CO2 and 90% N2 at
37.0 C for 7 days.
Treatment 2:mSOFaa under 5% O2, 5% CO2 and 90% N2 at 38.5
C for 2 days and HECM-9 under 5% O2, 5% CO2
and 90% N2 at 37.0 C for 5 days.
Treatment 3:mSOFaa under 5% O2, 5% CO2 and 90% N2 at 37.0
C for 7 days.
Treatment 4:mSOFaa under 5% O2, 5% CO2 and 90% N2 at 37.0
C for 2 days and HECM-9 under 5% O2, 5% CO2
and 90% N2 at 37.0 C for 5 days.
Treatment 5:mSOFaa under 5% O2 and 5% CO2 in air at 38.5 C
for 2 days and mSOFaa under 20% O2 and 5% CO2
in air at 38.5 C for 5 days.
Treatment 6:mSOFaa under 5% O2 and 5% CO2 in air at 38.5 C
for 7 days.
The embryos in Treatment 5 were co-cultured with bovine ovi-
ductal epithelial cells (BOVD) from day three to day seven [24,
38]. Bovine PA embryos were cultured in all treatments as the con-
trol. Bovine SCNT embryos were cultured in Treatment 5 and 6 as
the internal control.
Interspecies embryo examination and collection
Monkey iSCNT embryos were examined at the two-, four-, and
eight- cell, morula, early blastocyst, expanded blastocyst and
hatched blastocyst stages.

3. LORTHONGPANICH et al.308
Oct-4 protein distribution by immunocytochemical analysis
and embryonic cell count
Monkey iSCNT, bovine SCNT and bovine PA embryos at the
two-cell to hatched blastocyst stages were washed in PBS prior to
fixation in 4% paraformaldehyde (PFA) for 30 min at room temper-
ature. The embryos were then permeabilized with 0.2% Triton-X
and 0.1% Tween 20 and blocked with 10% normal goat serum
(NgS). They were then incubated overnight with Oct-4 (1:100) pri-
mary antibody (Chemicon International, Temecula, CA, USA).
After a thorough wash, the embryos were exposed to a secondary
antibody conjugated with FITC (1:1000; Chemicon International)
for 2 h. The embryos were counterstained with 2 μg/ml 4’-6-dia-
midino-2-phenylindole (DAPI) for 10 min, and individual embryos
were mounted on slides and subjected to fluorescent microscopic
examination.
Statistical analysis
For each treatment group, the experiments were replicated at
least three times. Data analyses for the differences in embryonic
development were carried out by ANOVA using the Statistical
Analysis System software (SAS, version 9.0; SAS, Cary, NC,
USA). For embryo development, the percentages of cleavage to
the blastocyst stage were calculated by dividing those numbers
with the number of cleaved embryos and analyzed by ANOVA
using the SAS software.
Results
Fusion rate of monkey fibroblasts with bovine enucleated
oocytes
The monkey fibroblasts were fused with enucleated bovine
oocytes using the same fusion parameters as bovine fibroblasts.
The fusion rates of monkey fibroblasts and bovine enucleated
oocytes were no different from that of bovine fibroblasts with
bovine enucleated oocytes, 157/195 (80.5 ± 8.5%) and 156/185
(84.3 ± 12.2%), respectively (Table 1). The reconstructed embryos
from the two groups were activated and cultured in mSOF media
containing 3 mg/ml BSA at 38.5 C in 5% O2, 5% CO2 and 90% N2
for two days to determine the cleavage rate. There was no differ-
ence in cleavage rate between monkey iSCNTs (143/157; 91.1 ±
7.5%) and bovine SCNTs (148/156; 94.9 ± 5.7%; Table 1). These
results suggested that (1) bovine fusion parameters are sufficient
for production of monkey iSCNT embryos and that (2) bovine oop-
lasm can promote dedifferentiation of monkey nuclei that is
comparable to that of bovine donor nuclei.
In vitro embryo development after culture in different media
The activated oocytes were cultured under six different culture
conditions. The developmental rates of the monkey iSCNT, bovine
SCNT and bovine PAs embryos are summarized in Table 2. The
cleavage rates were not significantly different between the monkey
iSCNT, bovine SCNT and bovine PA embryos (P≤0.05). How-
ever, the monkey iSCNT embryos cultured in Treatment 1 had the
lowest rate of development to the eight-cell stage compared with
the other treatments (P≤0.05). Significant differences among the
monkey iSCNT, bovine SCNT and bovine PA embryos were
observed on day four after reaching the 16-cell stage. The monkey
iSCNT embryonic cell nuclei could no longer divide nor increase in
number after the 16-cell stage; however, the cytoplasm could
divide further, and this was confirmed by DAPI staining. Thus, the
monkey iSCNT embryos resembled early blastocysts with unclear
blastocoels. After staining the monkey iSCNTs with DAPI, about
20% of the total embryonic cells showed DNA fragmentation,
which is a sign of apoptosis (n=5 embryos). We named this kind of
embryo an early blastocyst-like embryo (Fig. 1d). Comparison of
the six treatments indicated that HECM-9 and mSOFaa medium
could not support monkey iSCNT embryos development to the
blastocyst stage with normal embryonic cell nuclei as it did in
bovine SCNT and bovine PA embryos (Table 2). Although
HECM-9 has been widely used for monkey embryo culture, it may
not be optimal for monkey iSCNT embryos (Fig. 1).
Monkey iSCNT embryo developmental timeline
Our results showed that monkey iSCNT, bovine SCNT and
bovine PA embryos had similar developmental timelines up to the
morula stage. They reached the two-cell, eight-cell and morula
stages at 24, 48 and 96 h, respectively, but only bovine SCNT and
bovine PA embryos reached the blastocyst stage 168 h after in vitro
culture (Fig. 2), regardless of the different culture conditions. On
the other hand, monkey iSCNT embryos showed early blastocyst-
like morphology at 168 h. Interestingly, unlike bovine SCNT and
PA embryos, monkey iSCNT embryos did not achieve the compact
morula stage at 120 h after in vitro culture.
Table 1. Fusion and cleavage rates after electrofusion of monkey and bovine fibroblasts to enucleated bovine
oocytes
Donor cell type No. of experiment No. of oocytes Fusion rate Cleavage rate
(% ± SD) (% ± SD)
Monkey fibroblasts 3 195 157/195 143/157
(80.5 ± 8.5) (91.1 ± 7.5)
Bovine fibroblasts 3 185 156/185 148/156
(84.3 ± 12.2) (94.9 ± 5.7)
The fusion rate was calculated based on the number of fused cells divided by the number of embryos that passed
through the electric current generated by the fusion machine, and the cleavage rate was calculated from the number
of cleaved embryo divided by the number of fused embryos (P≥0.05).

4. 309MONKEY iSCNT DEVELOPMENT
Table 2. Development of iSCNT and SCNT embryos cultured under different conditions
Treatment No. Cleavage (%) 8-cell (%) Morula (%) Blastocyst (%)
iSCNT 102 93 48 36 20*
1. HECM-9 at 37.0 C (7D) (91.2) a
(47.1) b
(38.7) c,d
(21.5) c,d
Non co-culture PA 105 94 62 55 21
(89.5) a
(59.0) a
(58.5) a,b
(22.3) c,d
2. mSOFaa at 38.5 C (2D) + iSCNT 97 96 84 48 31*
HECM-9 at 37.0 C (5D) (99.0) a
(86.6)a
(50.0) b,c
(32.3) a,b,c
Non co-culture PA 105 88 60 29 17
(83.8) a
(57.1) a
(32.9) c,d
(19.3) c,d
iSCNT 92 91 68 37 33*
3. mSOFaa at 37.0 C (8D) (98.9) a
(73.9) a
(40.7) c,d
(36.3) a,b
Non co-culture PA 100 78 54 36 17
(78.0) a
(54.0) a
(46.2) b,c
(21.8) c,d
4. mSOFaa at 37.0 C (2D) + iSCNT 100 95 78 37 12*
HECM-9 at 37.0 C (5D) (95.0) a
(78.0) a
(38.9) c,d
(12.6) d
Non co-culture PA 105 85 65 46 19
(80.9) a
(61.9) a
(51.7) b,c
(21.3) c,d
iSCNT 110 104 93 62 18*
(94.5) a
(84.5) a
(59.6) a,b
(17.3) c,d
5. mSOFaa at 38.5 C (7D) SCNT 94 93 78 65 39
Co-culture (98.9) a
(83.0) a
(69.8) a
(41.9) a
PA 105 94 63 35 23
(89.5) a
(60.0) a
(37.2) c,d
(24.5) c,d
iSCNT 110 104 84 57 31*
(94.5) a
(76.4) a
(54.8) b,c
(29.8) b,c,d
6. mSOFaa at 38.5 C (7D) SCNT 120 108 94 40 13
Non co-culture (90.0) a
(78.3)a
(37.0) c,d
(12.0) d
PA 100 86 60 53 12
(86.0) a
(60.0) a
(57.6) b,c
(13.9) d
A–d: Different superscripts within the column indicate significant differences (P≤0.05), * Early blastocyst-like embryo.
Fig 1. Monkey iSCNT (a–d), bovine SCNT (e–h) and bovine PA (i–l)
embryo development at 24, 48, 72–96 and 168 h after in vitro
culture.
Fig 2. In vitro developmental timelines of the monkey iSCNT, bovine
SCNT and bovine PA embryos. All of the embryos are able to
develop with similar timelines even when they were cultured in
different culture systems. However, they had different blastocyst
rates. The bovine SCNT and bovine PA were able to hatch from
zonae pellucidae at 192 h, but development of the monkey iSCNT
embryos stopped at the early blastocyst-like stage 168 h after
culture.

5. LORTHONGPANICH et al.310
Embryonic cell number
Monkey iSCNT embryos cultured under different conditions
achieved similar cell numbers (Table 3). The total cell numbers of
the monkey iSCNT embryos at the early blastocyst stage were sig-
nificantly different from the bovine PA and bovine SCNT embryos
cultured under the same conditions (P≤0.01).
Oct-4 distribution in iSCNT, SCNT and PA embryos
The distribution of Oct-4 protein varied between the iSCNT,
SCNT and PA embryos. According to the developmental results,
the monkey iSCNT embryos were not capable of forming an
expanded blastocyst. Therefore, the distribution of Oct-4 in the
monkey iSCNT embryos was only determined from the two-cell
stage up to blastocyst-like stage (Fig. 3). Oct-4 was detected in
monkey iSCNT and bovine SCNT embryos at all stages and was
observed solely in the nuclei (Figs. 3 and 4). However, the Oct-4
expression in the bovine PA embryos was only detected at the
morula and later stages (Fig. 5). Both bovine SCNT and PA
embryos had higher levels of Oct-4 in the ICM cells of hatching
blastocysts (bovine SCNT; day seven after culture) and hatched
blastocysts (bovine PA; day eight after culture). We also observed
that expression of Oct-4 in the trophoblast cells was decreased in
the SCNT embryos after about 50% of the blastomeres hatched (n=
10; Fig. 4).
Discussion
We have demonstrated that monkey somatic cell nuclei could be
dedifferentiated in bovine ooplasm and that a cleavage rate and
developmental rate up to the eight-cell stage comparable to that of
bovine SCNT and bovine PA embryos could be achieved. HECM-
9 sufficiently supported bovine embryo development under a non
co-culture system. Embryo quality and cell number were also com-
parable to mSOFaa, which is commonly used in bovine. The
mSOFaa media provided better support when embryos were co-
cultured with BOVD. Different temperatures did not create an
enhancing effect on embryo development, and different culture
media did not enhance embryo growth, which was largely affected
by nuclear manipulation. Li et al. suggested that a closer genetic
background between the donor cell and recipient oocyte could
enhance blastocyst development in vitro [40]. This suggests that
the low blastocyst rate in the monkey iSCNTs might be due to the
genetic distance between bovine and higher primates, which may
result in incompatible genomic regulation and metabolic mecha-
nism. The birth of a live cloned gaur (Bos gaurus) by iSCNT [6]
and successful implantation of a yak (Bos grunniens) iSCNT
embryo [13] using bovine oocytes as recipient cytoplasm suggests
that a close phylogenetic distance between the donor nucleus and
recipient cytoplasm results in a higher likelihood of success in
delivering live offspring. In our study, the monkey iSCNT
embryos could not develop beyond the eight-cell stage, which was
consistent with previous reports concerning iSCNT mouse [37, 41],
dog [42] and camel [11] embryos using bovine [37, 41, 42] and
ovine [11] enucleated oocytes as the recipient cytoplasm. Zhou et
al. [12] have investigated reprogramming of the equine somatic
cell nucleus after injection into bovine enucleated oocyte cyto-
plasm and the developmental potential of the reconstructed
embryos. They found that only 17% (8/48) of the equine iSCNT
embryos could develop to the four- to eight-cell stage, and none of
them could develop to the blastocyst stage, while 15% (10/68) of
the bovine PA embryos (control group) reached the blastocyst
stage. These reports [11, 12, 37, 40, 41] suggest that the iSCNT
Table 3. The embryonic cells number after embryos were cultured in different culture conditions
Treatment Embryo type Number of embryos Cell number ± SD
1. HECM-9 at 37.0 C (7D) iSCNT 10 15.3 ± 2.2
Non co-culture PA 10 114.2 ± 11.7
2. mSOFaa at 38.5 C (2D) + iSCNT 10 13.2 ± 3.6
HECM-9 at 37.0 C (5D) PA 10 64.3 ± 22.8
Non co-culture
3. mSOFaa at 37.0 C (7D) iSCNT 10 14.2 ± 2.8
Non co-culture PA 10 84.7 ± 24.7
4. mSOFaa at 37.0 C (2D) + iSCNT 10 14.2 ± 2.3
HECM-9 at 37.0 C (5D) PA 10 99.5 ± 13.9
Non co-culture
5. mSOFaa at 38.5 C (7D) iSCNT 10 14.8 ± 2.2
Co-culture SCNT 10 146.9 ± 16.5
PA 10 128.2 ± 26.2
6. mSOFaa at 38.5 C (7D) iSCNT 10 14.5 ± 2.5
Non co-culture SCNT 10 105.7 ± 10.4
PA 10 125.4 ± 27.9
The cell numbers of the monkey iSCNT embryos from each treatment were significantly different from those
of the bovine PA and bovine SCNT embryos (P≤0.01); however, the bovine SCNT and bovine PA embryos
(Tr 5 and Tr 6) were not significantly different (P≥0.05).

6. 311MONKEY iSCNT DEVELOPMENT
embryos of phylogenetically unrelated donor cells and recipient
cytoplasm could undergo the first embryonic division and subse-
quently developed to the eight-cell and morula stages using bovine
or ovine oocytes as the recipient cytoplasm; however, these iSCNT
embryos were incapable of developing into blastocysts.
Meirelles et al. [43] suggested that maternal embryonic transi-
tion (MET) is related to the developmental block in early pre-
implantation embryos. However, the developmental block in the
iSCNT embryos in their study was largely unclear because the
iSCNT embryos were a hybrid of two different species. MET
occurs at the eight-cell stage in bovine embryos [30] and at the
four- to eight-cell stage in primate embryos [44, 45]. At MET, the
role of maternally inherited regulatory molecules is diminished as
the embryo develops and until the embryonic genome is fully acti-
vated; therefore, the MET in iSCNT embryos is expected to be
under the influence of the two species. Based on our findings, the
nuclear genome of the monkey iSCNT embryo is unlikely to acti-
vate fully and support development beyond the eight-cell stage.
Moreover, Park et al. [37] studied the nuclear remodeling of
iSCNT embryos using bovine enucleated oocytes as the recipient
cytoplasm for bovine and mouse donor nuclei. They found that the
embryos that received nuclei from bovine fibroblast cells devel-
oped into blastocysts and that received nuclei from mouse
fibroblasts did not develop beyond the eight-cell stage. They sug-
gested that a similar pattern of nuclear remodeling procedures were
observed in oocytes reconstructed with bovine and mouse fibro-
blast cells. However, the numbers of housekeeping mouse genes
(hsp70, bax and glt-1) were abnormally expressed, and mouse Oct-
4 mRNA could not be detected in the embryos that received mouse
nuclei. These results suggest that the mouse embryonic genome
did not receive sufficient activation from the SCNT procedures
Fig 3. The Oct-4 distributions of 2-cell (a–c), 8-cell (d–f), 16-cell (g–i)
and early blastocyst-like monkey iSCNT embryos (j–l). DAPI
and Oct-4 are shown in blue and green, respectively. Oct-4
could be detected in every nucleus of the monkey iSCNT
embryo. Bar=100 μm.
Fig 4. The Oct-4 distribution of 4-cell (a–c), hatching (d–f) and late-
hatching blastocyst stage bovine SCNT embryos (g–i). DAPI
and Oct-4 are shown in blue and green, respectively. Oct-4
expression could be detected in every nucleus of the bovine
SCNT embryo up to the early blastocyst stage, and the
expression decreased in TE cells when the embryo reached the
expanded blastocyst stage. The Oct-4 expression of the hatching
and hatched blastocysts are stronger in ICM cells than TE cells.
Bar=100 μm.
Fig 5. The Oct-4 distribution in bovine PA embryos. DAPI and Oct-4
are shown in blue and green, respectively. Oct-4 expression was
not detected in PA embryos before the four-cell stage (a–c).
However, it was detected in every embryonic cell at the morula
stage (d–f). The Oct-4 expression in the TE cells of the
blastocyst embryo decrease when the embryos increase the
expansion. Finally, the ICM cells show stronger Oct-4
expression compared with the very low expression in TE cells
(g–i). Bar=100 μm.

7. LORTHONGPANICH et al.312
[37]. Similarly, in the present study, the monkey iSCNT embryos
could not develop beyond the 16-cell stage, and this might be due
to inadequate embryonic genome activation. According to Betts
and King [46], there are two possible mechanisms that might have
resulted in a developmental block: (1) inability to overcome chro-
matin repression and activate transcription of important
developmental genes and/or (2) inability to respond to injuries
caused by environmental factors. We have shown that the bovine
PA and bovine SCNT embryos were able to develop to the blasto-
cyst stage, and this brings into question whether these mechanisms
were the reasons for embryonic arrest in our study. We expect that
the cause of embryonic arrest in the monkey iSCNT embryos is
likely alteration of the gene expression profile of important devel-
opmental related genes, such as Oct-4 and E-Cad genes. However,
further study is needed to confirm this.
Van Eijk et al. [32] studied Oct-4 protein expression in bovine
IVF derived embryos using immunocytochemical analysis. Their
results suggest that bovine IVF derived embryos show Oct-4 pro-
tein expression in all nuclei throughout all embryonic
developmental stages until day ten of development, which is con-
sistent with the bovine SCNT and monkey iSCNT embryos in the
present study. Our results showed that Oct-4 protein was detected
at the two-cell stage up to the blastocyst stage in bovine SCNT
embryos and up to the 16-cell stage in monkey iSCNT embryos,
but it was undetectable until the morula stage in bovine PA
embryos. So, it is possible that the Oct-4 protein in a monkey
iSCNT embryo comes from embryonic mRNA because the PA
embryo, which carried only maternal mRNA, did not show Oct-4
protein at an early stage of development.
Several studies have demonstrated the methylation status of the
Oct-4 promoter, which is highly correlated with the expression of
Oct-4. The Oct-4 promoter is activated when it is unmethylated,
which normally occurs in pre-implantation embryos. On the other
hand, Oct-4 promoter is methylated in differentiated cells [47–49].
Our results also showed that bovine PA embryos had Oct-4 expres-
sion patterns that were different from those of the cloned embryos.
Such differences might be the result of incomplete dedifferentiation
and reprogramming processes. Furthermore, the epigenetic status
of the donor cell also plays an important role in the reprogramming
process [50]. Therefore, in-depth investigation in these areas will
further clarify our findings.
In conclusion, we have demonstrated that a monkey somatic cell
nucleus could be dedifferentiated after reconstruction with bovine
cytoplasm. However, incomplete reprogramming in the monkey
iSCNT embryos resulted in embryonic arrest at the eight-cell stage.
The culture conditions have no significant effect on iSCNT embryo
development, and the expression patterns of Oct-4 in cloned
embryos are different from that of the PA embryo.
Acknowledgements
The authors would like to thank Mr. S Imsoonthronruksa, Ms. K
Srirattana, C Tangthai and N Sripanya (Embryo Technology and
Stem Cell Research Center, Suranaree of University of Technol-
ogy, Nakhon Ratchasima, Thailand) for technical assistance and
Mrs. L. Sinclair for critical review of this study. We would also
like to express our gratitude to the Nakhon Ratchasima Zoo for pro-
viding the monkey skin tissues that were used as donor cells in this
experiment. This study was supported by The Royal Golden Jubi-
lee Ph.D. Program of the Thailand Research Fund.
References
1. Byrne JA, Pedersen DA, Clepper LL, Nelson M, Sanger WG, Gokhale S, Wolf DP,
Mitalipov SM. Producing primate embryonic stem cells by somatic cell nuclear trans-
fer. Nature 2007; 450: 497–505.
2. Chen DY, Sun QY, Liu JL, Li GP, Lian L, Wang MK, Han ZM, Song XF, Li JS, Sun Q,
Chen YC, Zhang YP, Ding B. The giant panda (Ailuropoda melanoleuca) somatic
nucleus can dedifferentiate in rabbit ooplasm and support early development of the
reconstructed egg. Sci China (Series C) 1999; 29: 324–330.
3. Chen DY, Wen DC, Zhang YP, Sun QY, Han ZM, Liu ZH, Shi P, Li JS, Xiangyu JG,
Lian L, Kou ZH, Wu YQ, ChenYC, Wang PY, Zhang HM. Interspecies implantation
and mitochondria fate of panda-rabbit cloned embryos. Biol Reprod 2002; 67: 637–642.
4. Dominko T, Mitalipova M, Haley B, Beyhan Z, Memili E, Mckusick B, First NL.
Bovine oocyte cytoplasm supports development of embryos produced by nuclear
transfer of somatic cell nuclei from various mammalian species. Biol Reprod 1999; 60:
1496–1502.
5. White KL, Bunch TD, Mitalipov S, Reed WA. Establishment of pregnancy after the
transfer of nuclear transfer embryos produced from the fusion of Argali (Ovis ammon)
nuclei into domestic sheep (Ovis aries) enucleated oocytes. Cloning 1999; 1: 47–54.
6. Lanza RP, Cibelli J, Diaz F, Moraes C, Farin PW, Farin CE, Hammer CJ, West MD,
Damiani P. Cloning of an endangered species using interspecies nuclear transfer.
Cloning 2000; 2: 79–90.
7. Lorthongpanich C, Laowtammathron C, Muenthaisong S, Vetchayan T, Ketudat-
Cairns M, Likitdecharote B, Parnpai R. In vitro development of enucleated domestic
cat oocytes reconstructed with skin fibroblasts of domestic and leopard cats. Reprod
Fert Dev 2004; 16: 149 (abstract).
8. Thongphakdee A, Numchaisrika P, Omsongkram S, Chatdarong K, Kamolnor-
ranath S, Dumnui S. Techakumphu M. In vitro development of marbled cat embryos
derived from interspecies somatic cell nuclear transfer. Reprod Dom Anim 2006; 41:
219–226.
9. Marshall A. Cloning for conservation. Nat Biotechnol 2000; 18: 1129.
10. Loi P, Ptak G, Barboni B, Fulka J, Cappai P, Clinton M. Genetic rescue of an endan-
gered mammal by cross-species nuclear transfer using post-mortem somatic cells. Nat
Biotechnol 2001; 19: 962–964.
11. Zhou H, Guo Z. Heterogeneous nuclear-transferred embryos reconstructed with
camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their
development H-M. Anim Reprod Sci 2006; 95: 324–330.
12. Zhou H, Liu C, Wang W. Heterospecific nuclear-transferred embryos derived from
equine fibroblast cells and enucleated bovine oocytes. Reprod Dom Anim 2007; 42: 243–
247.
13. Kittiyanant Y, Saikhun J, Chaisalee B, White KL, Pavasuthipaisit K. Somatic cell
cloning in buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and
activation procedures. Clon Stem Cells 2001; 3: 97–104.
14. Gomez M, Pope CE, Giraldo A, Lyons LA, Harris LA, King AL, Cole A, Godke RA,
Dresser BL. Birth of African wildcat cloned kittens born from domestic cats. Clon Stem
Cells 2004; 6: 247–258.
15. Li Y, Li S, Dai Y, Du W, Zhao C, Wang L, Eang H, Li R, Liu Y, Wan R, Li N. Nuclear
reprogramming in embryos generated by the transfer of yak (Bos grunniens) nuclei
into bovine oocytes and comparison with bovine-bovine SCNT and bovine IVF
embryos. Theriogenology 2007; 67: 1331–1338.
16. Ikumi S, Sawai K, Takeuchi Y, Iwayama H, Ishikawa H, Ohsumi S, Fukui Y. Inter-
species somatic cell nuclear transfer for in vitro production of Antarctic minke whale
(Balaenoptera bonaerensis) embryos. Clon Stem Cells 2004; 6: 284–293.
17. Peter JK, Milliken G, Davis DL. Development of porcine embryos in vitro: Effects of
culture medium and donor age. J Anim Sci 2001; 79: 1578–1583.
18. Lee JW, Wu SC, Tian XC, Barber M, Hoagland T, Riesen J, Lee KH, Tu DF, Cheng W
TK, Yang X. Production of cloned pigs by whole-cell intracytoplasmic microinjection.
Biol Reprod 2003; 69: 995–1001.
19. Wakayama T, Rodriguez I, Perry ACF, Yanagimachi R, Mombaerts P. Mice cloned
from embryonic stem cells. PNAS USA 1999; 96: 14984–14989.
20. Heindryckx B, Rybouchkin A, Elst VDJ, Dhont M. Effect of culture media on in vitro
development of cloned mouse embryos. Cloning 2001; 3: 41–50.
21. Wakayama S, Ohta H, Kishigami S, Thuan NV, Hikichi T, Mizutani E, Miyake M,
Wakayama T. Establishment of male and female nuclear transfer embryonic stem cell
lines from different mouse strains and tissue. Biol Reprod 2005; 72: 932–936.

8. 313MONKEY iSCNT DEVELOPMENT
22. Nagy A, Gertsenstein M, Vintersten K, Behringer R. In: Manipulating the Mouse
Embryo: A Laboratory Manual, 3rd
ed, New York: Cold Spring Harbor Laboratory
Press, 2003.
23. Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and develop-
ment for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum
and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate
development. Biol Reprod 1994; 50: 390–400.
24. Laowtammathron C, Lorthongpanich C, Ketudat-Cairns M, Hochi S, Parnpai R. Fac-
tors effecting cryosurvival of nuclear-transferred bovine and swamp buffalo blasto-
cysts: the effects of hatching stage, linoleic acid albumin in culture medium and ficoll
supplementation solution. Theriogenology 2005; 64: 1185–1196.
25. Mello MRB, Ferguson CE, Lima AS, Wheeler MB. In vitro development of bovine
embryos cultured in KSOM, CR1aa or KSOM/CR1aa. Reprod Fertil Dev 2005; 17: 221–
222.
26. Zhang P, Patal B, McMenamin M, Reddy SE, Paprocki AM, Schramm RD, Latham
K. The primate embryo gene expression resource: A novel resource to facilitate rapid
analysis of gene expression patterns in non-human primate oocytes and preimplan-
taion stage embryos. Biol Reprod 2004; 70: 1411–1418.
27. Mitalipov SM, Kuo HC, Hennebold JD, Wolf DP. Oct-4 expression in pluripotent
cells of the rhesus monkey. Biol Reprod 2003; 69: 1785–1792.
28. Schramm RD, Paprocki AM, VandeVoort CA. Causes of developmental failure of in-
vitro matured rhesus monkey oocytes: Impairments in embryonic genome activation.
Hum Reprod 2003; 18: 826–833.
29. Hoffert KA, Anderson GB, Wildt DE, Roth TL. Transition from maternal to embry-
onic control of development in IVM/IVF domestic cat embryos. Mol Reprod Dev 1997;
48: 208–215.
30. Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control
in early mammalian development: a comparison of several species. Mol Reprod Dev
1990; 26: 90–100.
31. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I,
Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo
depends on the POU transcription factor Oct4. Cell 1998; 95: 379–391.
32. Van Eijk MJT, Rooijen VMA, Modina S, Scesi L, Folkers G, Tol VHTA, Bevers MM,
Fisher SR, Lewin HA, Rekacolli D, Galli C, Vaureix C, Trounson AO, Mummery
CL, Gandolfi F. Molecular cloning, genetic mapping, and developmental expression
of bovine POU5F1. Biol Reprod 1999; 60: 1093–1103.
33. Kirchhof N, Carnwath JW, Lemme E, Anastassiadis K, Scholer H, Niemann H.
Expression pattern of Oct-4 in preimplantation embryos of different species. Biol
Reprod 2000; 63: 1698–1705.
34. Kurosaka S, Eckardt S, McLaughlin KJ. Pluripotent lineage definition in bovine
embryos by Oct-4 transcript localization. Biol Reprod 2004; 71: 1578–1582.
35. Wang L, Duan E, Sung LY, Jeong BS, Yang X, Tian XC. Generation and characteriza-
tion of pluripotent stem cells from cloned bovine embryos. Biol Reprod 2005; 73: 149–
155.
36. Lorthongpanich C, Srirattana K, Imsunthornruksa S, Sripunya N, Laowtammathron
C, Kumpong O, Ketudat-Cairns M, Parnpai R. Expression and distribution of Oct-4
in interspecies cloned long tailed monkey (Macaca fascicularis) embryos. Reprod Fert
Dev 2007; 19: 149.
37. Park SH, Shin MR, Kim NH. Bovine oocyte cytoplasm supports nuclear remodeling
but not reprogramming of murine fibroblast cells. Mol Reprod Dev 2004; 68: 25–34.
38. Parnpai R, Tasripoo K, Kamonpatana M. Development of cloned swamp buffalo
embryos derived from fetal fibroblasts: comparison in vitro cultured with or without
buffalo and cattle oviductal epithelial cells. Buffalo J 1999; 15: 371–384.
39. Zimmermann U, Vienken J. Electric field-induced cell-to-cell fusion. J Membr Biol
1982; 67: 165–182.
40. Li Y, Dai Y, Du W, Zhao C, Wang H, Wang L, Li R, Liu Y, Wan R, Li N. Cloned
endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and
comparison of the blastocyst development with yak (Bos grunniens) and bovine. Mol
Reprod Dev 2006; 73: 189–195.
41. Arat S, Rzucidlo SJ, Stice SL. Gene expression and in vitro development of inter-spe-
cies nuclear transfer embryos. Mol Reprod Dev 2003; 66: 334–342.
42. Murakami M, Otoni T, Wongsrikeao P, Agung B, Sambuu R, Suzuki T. Develop-
ment of interspecies cloned embryos in yak and dog. Clon Stem Cells 2005; 77–81.
43. Meirelles FV, Caetano AR, Watanabe YF, Ripamonte P, Sarambula SF, Merighe GK,
Garcia SM. Genome activation and developmental block in bovine embryos. Anim
Reprod Sci 2004; 82: 13–20.
44. Artley JK, Braude PR, Johnson MH. Gene activity and cleavage arrest in human pre-
embryos. Hum Reprod 1992; 7: 1014–1021.
45. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four-
and eight-cell stages of preimplantation development. Nature 1988; 332: 459–461.
46. Betts DH, King WA. Genetic regulation of embryo death and senescence. Theriogenol-
ogy 2001; 55: 171–191.
47. Gidekel S, Bergman Y. A unique developmental pattern of Oct-3/4 DNA methylation
is controlled by a cis-demodification element. J Biol Chem 2002; 277: 34521–34530.
48. Hattori N, Nishino K, Ko YG, Hattori N, Ohgane J, Tanaka S, Shiota K. Epigenetic
control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem
cells. J Biol Chem 2004; 279: 17063–17069.
49. Simonsson S, Gurdon J. DNA demethylation is necessary for the epigenetic repro-
gramming of somatic cell nuclei. Nat Cell Biol 2004; 6: 984–990.
50. Blelloch R, Wang Z, Meissner A, Pollard S, Smith A, Jaenisch R. Reprogramming
efficiency following somatic cell nuclear transfer is influenced by the differentiation
and methylation state of the donor nucleus. Stem Cells 2006; 24: 2007–2013.