Proteomic Analysis in the Cloned Porcine Conceptuses
at Day 14 of Pregnancy
Yeoung-Gyu Ko, Hyun-mi Kim, Byoung-Chul Yang, Hwi-Cheul Lee, Seongsoo Hwang, Gi-Sun Im, Keon-Bong Oh, Mi-Rung Park, Jae-Seok Woo and
Soo-Bong Park; National Institute of Animal Science, RDA, Namwon 590-832, Korea
ABSTRACT: Although cloned animals have been successfully produced, a very low birth rate by SCNT embryos has been observed. Such low efficiency of cloning could possibly
arise from abnormal or poorly developed placenta and fetus. In the present study, we present differentially regulated proteins in the conceptuses from SCNT embryos to understand the
molecular nature of the tissue. Proteins expressed specifically or prominently on day 14 were identified in cloned porcine conceptuses using two-dimensional polyacrylamide gel
electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The conceptuses were obtained from the recipients pregnant by
natural mating (control) or SCNT embryos, respectively. The size and morphology of the samples looked similar in both groups. In the proteomic analysis, 67 proteins were identified as
differentially regulated proteins in the SCNT conceptuses. Among these proteins, 61 were down-regulated, whereas the other 6 proteins were up-regulated. In the down-regulated
proteins, 2 elongation factors and 5 isomerase were considered to be involved in regulation of early-implantation development and peroxiredoxin-4, Oxidation resistance 1 and
thioredoxin peroxidase II, which is antioxidant enzymes reduce oxidative stress by scavenging reactive oxygen species (ROS). Among the up-regulated proteins, annexin V, an apoptotic
regulator that interacts with the key components of apoptotic signaling, was identified. These findings demonstrate that the conceptuses from SCNT embryos showed aberrant protein
expression patterns during pre-implantation development; although the morphology of the tissues looked normal, and it can be suggested that the abnormal function of conceptuses in
clone might be related negatively to placental formation and fetal development. (Key words : 2-DE, MALDI-TOF, Cloned porcine Conceptus, )
Material and Methods
Table2. Down-regulated proteins in the SCNT conceptuses.
Collection of extraembryonic tissue: In vitro maturation, nuclear transfer, and embryo
culture were performed according to previously reports (Hwang et al., 2008). Somatic cells
were the fetal fibroblasts isolated from 35 day fetus. Approximately, 200 SCNT embryos were
transferred to the oviducts of each synchronized recipient. The conceptuses from SCNT
embryos and three control at the 14 day of pregnancy were prepared.
Two-dimensional gel electrophories (2DE) proteomic analysis: 2-DE was performed as
descrived previously (Lee et al., 2006). IEF was carried out with the IPGphor (Genomine Corp.,
Pohang, Korea) using precast 24-cm pH 4-10 nonlinear IPG gel strips. The second dimension
separation was carried out using 12% SDS-PAGE gels. Electrophoresis was repeated three
times in each sample to ensure reproducibility. Silver staining kit was used for 2-DE gels.
Proteomic analysis: Silver stained gels were scanned with imageScanner and analysed with
PD-Quest software (version 7.0, BioRad). Destaining and in-gel trypsin digestion of protein
spots were performed as described previously (O’Neil et al.,2002). In-gel digested peptides
were analyzed with an Ettan MALDI-TOF(GE Healthcare Bio-Sciences Corp.). Mass spectras
were calibrated and processed using Flex Analysis and ProFound search engine.
Results
A
B
C
D
E
F
SCNT
NM
Spot
no.
2309
3104
3702
3808
4103
4202
4207
4209
4303
4309
4507 Glutamine-dependent asparagine synthetase
4703 RuvB-like protein 2
4808 Aldehyde oxidase, Retinal oxidase
5002
5004
5005
5006
5007
5103
5105
5106
5204
5305
5402
5404
5502
5604
5702
5706
5708
5802
5805
5806
6002
6004
6007
6012
6201
6302
6404
6405
6501
6504
6509
6506
6807
7002
7203
7205
7301
7506
Fig. 1. Two-dimensional gel electrophoresis. Protein were isolated from normal and SCNT
conceptuses at day 14 of pregnancy, and 600
of total protein were loaded to the 2-DE gel.
The first Dimension was 24-cm pH 4-10 nonlinear IPG, and the second dimension was
10~16% gradient gels (A, B and C, SCNT; D, E and F , Natural mating (conceptus). The
proteins were visualized by the by the silver staining. Among 800 spots, 116 spots were
selected.
8001
8003
8004
8104
8106
8111
8201
8303
8310
8602
8801
Fig. 2. Selected spot images of 2-D gel. Up-regulated spots in SCNT conceptus : 9 spots
Down-regulated Spots spots in SCNT conceptus : 107 spots.
Protein name
Beta-acti
Fasciculation and elongation protein zeta 2 splicing
variant 2
Similar to ATPase, H+ transporting, lysosomal 70kD
Valosin-containing protein
ACTG1 protein
Pyruvate dehydrogenase:SUBUNIT=beta
Dehydrogenase H4,lactate
F-actin capping protein alpha-1 subunit
Crystal Structure of Bovine Retinal Creatine Kinase
Creatine kinase B chain
Ubiquitin
Neuron specific gene family member 2
Cystatin-B
Cytochrome P450
Testis specific ankyrin-like protein 1
Annexin-4
Laminin-binding protein
Dehydrogenase (ubiquinone) Fe-S protein 3
TALDO1 protein
Translation initiation factor
ARP3 actin-related protein 3 homolog
protein disulfide isomerase-related protein 5
Coronin, actin binding protein, 1B isoform 4
Chaperonin containing TCP1, subunit 5 (epsilon)
Cad protein
Neurofascin homolog (chicken)
Gelsolin
Metavinculin
Neutral alpha-glucosidase AB
Vinculin
Fatty acid binding protein 3
Retinol-binding protein 4
peroxiredoxin 4
Ferritin L subunit
Heat shock protein 90 beta
CUB and sushi domain-containing protein 3
p38-2G4
Guanosine diphosphate dissociation inhibitor 2
ADP-ribosylation factor-like 14
ER-60 protease
Oxidation registance 1
Coiled-coil domain containing 96
Thrombospondin 1
Phosphoglycerate mutase 1 (brain)
Chain A, Novel Nadph-Binding Domain Revealed
By The Crystal Structure Of Aldose Reductase
Malate dehydrogenase
Serine proteinase inhibitor, clade B, member 5
AAH07643 Similar to singed (Drosophila)-like
B Chain B, Ligand-Free Heterodimeric Human
Glutathione S-Transferase M2-3
Thioredoxin peroxidase II
Tpi1 protein
AAH09454 Unknown
Eef2 protein (elongation factor-2)
Elongation factor 2
Galactose mutarotase (aldose 1-epimerase )
Isocitrate dehydrogenase 1 (NADP+), soluble
A Chain A, Crystal Structure Analysis And Citrate
Synthase With Oxaloacetate And S-AcetonylCoenzyme A
Tubgcp6 protein
Aconitase 1
NCBI accession
Species
no.
CAA27396.1
Mus musculus
BAD06208.1
Rattus norvegicus
XP_545103
NP_999445.1
AAH23548.1
PRF: 448581
PRF: 229620
NP_006126.1
PDB: 1G0WA
spP05124.1
Canis familiaris
Sus scrofa
Homo sapiens
Sus scrofa
Homo sapiens
Bos taurus
Canis familiaris
Mesocricetus
spP17714.3
auratus
NP_035434.1
Mus musculus
Oryctolagus
spP80456.2
cuniculus
BAA11842.1
Cavia porcellus
NP_032767.1
Mus musculus
spQ29290.1
Sus scrofa
NP_898898.1
Homo sapiens
BAA92844
Homo sapiens
spP08132.2
Sus scrofa
CAA43469
Homo sapiens
NP_777244.1
Bos taurus]
AAH18847.2
Homo sapiens
CAA56074.1
Homo sapiens
NP_776651.1
Bos taurus
AAB50217.1
Homo sapiens
XP_508596.1
Pan troglodytes
NP_001004078.1 Rattus norvegicus
AAH53097.1
Mus musculus
CAI14661.1
Homo sapiens
CAA32077.1
Sus scrofa
spP26234.4
Sus scrofa
NP_999069.1
Sus scrofa
NP_999099.1
Sus scrofa
AAT72764.1
Sus scrofa
spP27485.2
Sus scrofa
NP_776858.1
Bos taurus
AAG16228.1
Sus scrofa
CAC84136.1
Bos taurus
spQ7Z407.2
Homo sapiens
CAA59260.1
Mus musculus
NP_001001643.1
Sus scrofa
AAH34354.1
Homo sapiens
BAA11928.1
Homo sapiens
AAH32710.1
Homo sapiens
NP_699207.1
Homo sapiens
NP_776621.1
Bos taurus
AAH62302.1
Homo sapiens
Matched
peptides
25
9
40
20
23
29
16
22
22
31
PMF (MS)
Measured
sequence
peptides coverage (%)
15
35
7
16
Experimental
Molecular
pI
mass (kDa)
39.45
5.8
39.41
4.5
19
16
10
12
10
8
13
18
33
23
49
29
28
41
36
43
68.78
89.96
29.68
36.11
36.72
33.08
42.85
42.97
5.3
5.1
5.5
5.4
6.7
5.4
5.5
5.5
11
7
14
64.83
6.1
14
13
30
51.27
5.5
12
9
8
149.29
6.3
11
6
8
7
7
32
22
24
9
8
24
8
10
8
8
9
5
27
43
6
11
2
20
5
21
14
15
31
12
27
7
9
8
12
7
4
5
5
7
20
8
8
7
7
19
6
8
8
7
8
5
18
28
6
7
8
6
5
10
8
10
19
6
14
7
8
6
11
24
27
60
12
11
45
32
23
14
14
40
20
11
16
7
8
8
19
27
6
58
42
30
48
49
3
29
40
32
31
13
17
7
55
34.97
19.15
11.17
62.43
107.17
36.04
31.89
30.38
37.56
47.25
47.76
46.52
54.90
59.98
112.04
94.97
85.1
124.5
107.16
117.43
13.27
23.39
30.72
18.31
27.95
409.84
38.24
50.76
21.89
57.18
85.45
62.98
133.51
28.92
6.9
9.6
5.9
9.0
6.3
5.7
4.8
6.3
6.4
6.3
5.5
5.0
5.6
5.5
6.1
9.0
5.9
5.6
5.6
5.9
6.9
5.4
6.0
5.8
7.1
5.5
8.3
6.3
9.3
6.0
5.0
4.9
4.7
6.7
PDB: 1DLAA
Sus scrofa
22
11
35
35.99
5.8
AAA31072.1
NP_033283.1
AAH07643.1
Sus scrofa
Mus musculus
Homo sapiens
21
7
22
10
6
9
35
20
25
31.98
42.49
46.21
6.1
5.5
6.6
PDB: 3GTUB
Homo sapiens
8
7
29
26.87
5.4
AAF32369.1
AAH61781.1
AAH09454
AAH02233.1
AAA37537.1
NP_999571.1
NP_005887.2
Cricetulus griseus
Rattus norvegicus
Homo sapiens
Mus musculus
Mus musculus
Sus scrofa
Homo sapiens
6
8
5
31
15
15
33
4
7
5
14
7
9
18
22
33
12
46
32
36
36
22.53
27.21
43.47
32.38
30.21
38.03
46.93
8.5
7.1
6.1
6.4
6.2
6.3
6.5
PDB: 4CTSA
Sus scrofa
28
16
33
49.13
7.0
AAH57626.1
NP_002188.1
Mus musculus
Homo sapiens
30
36
11
14
17
12
94.37
99.02
5.7
6.1
Fig. 3. The identified proteins organized according to molecular function.
Conclusions
Protein name
Spot no.
PMF (MS)
NCBI accession
no.
Species
Matched
peptides
Measured
peptides
sequence
coverage (%)
25
15
35
CAA27396.1
Mus musculus
3104
Beta-acti
Fasciculation and elongation protein zeta 2 splicing
variant 2
BAD06208.1
Rattus norvegicus
9
7
16
3702
Similar to ATPase, H+ transporting, lysosomal 70kD
XP_545103
Canis familiaris
40
19
33
3808
Valosin-containing protein
NP_999445.1
Sus scrofa
20
16
23
4103
ACTG1 protein
AAH23548.1
Homo sapiens
23
10
49
4202
Pyruvate dehydrogenase:SUBUNIT=beta
PRF: 448581
Sus scrofa
29
12
29
2309
Table1. Up-regulated proteins in the SCNT conceptuses.
In the proteomic analysis, 67 proteins were identified as differentially regulated proteins in the
SCNT conceptuses on day 14 .
Among the up-regulated proteins, antioxidant enzymes which are reduce oxidative stress by
scavenging reactive oxygen species (ROS) were considered to be involved in regulation of
early-implantation development.
These findings demonstrate that the conceptuses from SCNT embryos showed aberrant protein
expression patterns during pre-implantation development.