Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death
- Jong-Yi Park†1, 3,
- Jae-Hwan Kim†2,
- Yun-Jung Choi1,
- Kyu-Chan Hwang1,
- Seong-Keun Cho3,
- Ho-Hyun Park4,
- Seung-Sam Paik4,
- Teoan Kim5,
- ChanKyu Park1,
- Hoon Taek Lee1,
- Han Geuk Seo6,
- Soo-Bong Park7,
- Seongsoo Hwang7 and
- Jin-Hoi Kim1Email author
© Park et al; licensee BioMed Central Ltd. 2009
Received: 09 June 2009
Accepted: 05 November 2009
Published: 05 November 2009
Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets.
Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC.
These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development.
In the past decade, several species of animal, including goat, pig, sheep and cattle, have been cloned using scNT techniques . However, while cloning is widely used in basic research, as well as in some biomedical and agricultural applications, a number of substantial problems exist with the current technologies, including relatively low success rates and severe defects of the fetus and/or placenta resulting in abortion, neonatal death and postnatal disease [2–4]. It has been suggested that a major cause of early fetal loss of cloned animals is the high incidence of placental abnormalities that can occur throughout the entire gestation period.
Studies have shown that aberrant methylation in the trophetoderm of cloned blastocysts can induce global gene dysregulation in extra-embryonic regions, and that this type of gene dysregulation can potentially lead to the development of a dysfunctional placenta and have a detrimental effect on overall fetal development [5–7]. It has long been recognized that the major limitation of animal cloning with regard to efficient animal production relates to inadequate conceptus--maternal interactions through the placenta . This inadequacy leads to deficiencies in fetal growth in utero, which can affect the survival, health and well-being of the newborn and the productivity of the animal at adulthood. Inadequate conceptus-maternal interaction is particularly acute in animal production using scNT. The incidence of abnormal placental development is very high in scNT-derived animals, and is largely responsible for the frequent fetal loss, late-term pregnancy complications and perinatal mortality associated with this technique . Recently, we were able to successfully clone piglets using scNT, but the procedure resulted in high level of phenotypic abnormalities that compromised fetal and postnatal health [3, 7, 10, 11]. As a result, the birth rate and postnatal survival of the cloned piglets was very low. Subsequently, we determined that the low birth rate of the cloned animals was due largely to abnormal levels of apoptosis in extraembryonic tissue during early pregnancy [3, 12] and/or placental insufficiency at term .
A number of human umbilical cord malformations have been documented, most of which involve blood vessels . One such arterial malformation, human single umbilical artery (SUA) syndrome, in which one of the two umbilical arteries is absent in the cord, is believed to be caused by insufficient irrigation of the terminal portion of the embryo. SUA syndrome affects 0.2-1% of pregnancies, and the clinical symptoms vary depending on the stage of development at which it occurs . Hypoplasia of one of the two umbilical arteries also occurs, albeit much less frequently (0.03% of pregnancies). Hypoplastic umbilical arteries (HUAs) are defined as malformed arteries that exhibit an artery-artery diameter difference of approximately 50%. HUAs are associated with pre/perinatal morbidity and congenital abnormalities, and have also been linked to intrauterine growth retardation, maternal diabetes, polyhydramnios and congenital cardiopathy . The development of HUAs most likely represents an incomplete form of SUA syndrome .
Of the 65 term piglets generated by scNT, we observed the high incidence of umbilical cord malformation (9/65, 13.9%), which led us to speculate that piglets with MUC might be a good model for studying human HUA syndrome. In the current study, we demonstrated that scNT piglets with MUC exhibit impaired endothelial cell migration and angiogenesis, defects that might underlie the development of HUA and/or placental insufficiency in these animals. We also analyzed gene and protein expression patterns in the umbilical cord to identify putative umbilical cord markers (either proteins or protein modifications) that were associated with poor outcomes for the fetus. Using this approach, we can begin to elucidate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and to investigate the molecular mechanisms of angiogenesis during umbilical cord development.
Animals were maintained and experiments were conducted in accordance with the Kon-Kuk University Guide for the Care and Use of Laboratory Animals.
Somatic cell nuclear transfer and embryo transfer
Nuclear transfer was carried out as described in previous reports [10, 11, 17]. Briefly, the matured eggs with the first polar body were cultured in medium supplemented with 0.4 mg/ml demecolcine (Sigma) and 0.05 mol/l sucrose for 1 hour (hr). Sucrose was used to enlarge the perivitelline space of the eggs. Treated eggs with a protruding membrane were moved to medium supplemented with 5 mg/ml cytochalasin B and 0.4 mg/ml demecolcine and the protrusion was removed with a beveled pipette. A single donor fetal fibroblast cell derived from gestational day 30-derived Duroc, Berkshire and 3 way hybrid (Landrace × Duroc × Yorkshire) fetus was injected into the perivitelline space of each egg and electrically fused using two direct current pulses of 150 V/mm for 50 msec in 0.28 mol/L mannitol supplemented with 0.1 mM MgSO4 and 0.01% polyvinyl alcohol (Sigma). Fused eggs were cultured in medium with 0.4 mg/ml colcemid for 1 h before parthenogenetic activation, and then cultured in 5 mg/ml of CB-supplemented medium for 4 h. The reconstructed oocytes were activated by 2 direct current pulses of 100 V/mm for 20 msec in 0.28 mol/L mannitol supplemented with 0.1 mmol/L MgSO4, and 0.05 mmol/L CaCl2. Activated eggs were cultured in the medium for 6 days in an atmosphere of 5% CO2 and 95% air at 39°C.
Gilts (Duroc × Yorkshire × Landrace) of at least eight months of age were used as recipients. Estrus synchronization of recipients was carried out as reported previously [10, 11]. ScNT embryos were surgically transferred into oviducts of synchronized recipients. The pregnancy status of recipients was determined by ultrasound between days 30-35. Recipients produced scNT-derived piglets via vaginal delivery.
Umbilical cord samples obtained from three control piglets and 65 scNT-derived piglets were washed with PBS and used for the purification of endothelial cells or analysis of gene and protein expression. scNT-N samples with normal phenotype after 6 month of birth were selected by comparing with control groups and used for further experiments.
Isolation and characterization of porcine umbilical vein-derived endothelial cells (PUVEC)
PUVEC were obtained from MUC (designated as scNT-MUC) and normal (designated as scNT-N) umbilical cords from scNT piglets, or normal umbilical cords from piglets derived by artificial insemination (designed as control), as described previously . PUVEC were grown until they reached confluence (between 3 to 5 days), and were re-fed every 2 to 3 days by exchanging half of the growth medium with fresh medium. Alternatively, PUVEC were stored in liquid nitrogen until use. The purity of each PUVEC population was confirmed by immunostaining with an anti-PECAM-1 (CD31) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and counterstaining with 4',6-diamidino-2-phenylindole (DAPI; 1:15,000, Sigma, St. Louis, MO).
RNA isolation and real time reverse transcriptase (RT)-PCR
Total RNA was extracted from umbilical cord tissue using a Micro-to-Midi Total RNA Purification System (Life Technologies Inc., Carlsbad, CA, USA). Real-time RT-PCR was conducted using a DNA Engine OPTICON2 system (MJ Research, San Francisco, CA, USA) and SYBR Green as the double-stranded DNA-specific fluorescent dye (SYBR Green qPCR premix, FINNZYMES, Woburn, MA, USA). Target gene expression levels were normalized to tubulin gene expression, which was unaffected in scNT-derived pigs. The RT-PCR primer sets are shown in Additional file 1. Real-time RT-PCR was independently performed in triplicate for different samples and the data were expressed as the mean value of gene expression measured in individual control, scNT-N and scN-MUC, using each individual animal as experimental unit.
Endothelial cell (EC) migration/motility assay
Migration assays were performed as described by Rudolph et al , with slight modifications. PUVEC were grown in 35 mm culture dishes to 80% confluence. A wound was formed by clearing the monolayer from an area of the dish with a 200 μl pipette tip. The boundary of the wound was marked and the cells were allowed to incubate for 24 hr. The cells were fixed, stained with a 1:10 dilution of Giemsa (Sigma), and then photographed. Cell migration was measured by counting the number of cells that migrated into the cleared area of the dish. Data represents the means of four different random fields.
Tubule formation assay
Matrigel-sandwich tubule formation assays were performed as described previously [16, 20]. Briefly, cold Matrigel solution was added to a 96-well plate and allowed to air-dry. Following rehydration of the Matrigel, ECs derived from control, scNT-N or scNT-MUC were seeded onto the Matrigel at a density of 10,000 cells per well, and then allowed to incubate at 37°C for 24 hr. Microtubules were visualized by microscopy and photographed using a digital camera.
2-dimensional gel electrophoresis (2-DE)
2-DE analysis was performed by using 3 controls, 3 scNT-N, and 6 scNT-MUC samples with three replicates independently. Umbilical cords were solubilized in lysis buffer containing 7 M urea, 2 M thiourea, 4% w/v CHAPS, 40 mM DTT and 0.5% Pharmalyte pH 4-7. Insoluble material was removed by centrifugation. IPG strips (17 cm, pH 4-7, Bio-Rad, Hercules, CA, USA) were rehydrated overnight in 300 μL of lysate containing 500 μg of protein. Isoelectric focusing was performed using a Protein IEF Cell (Bio-Rad). The focused strips were then equilibrated by incubating them first in equilibration solution (6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris-Hcl, pH 8.8) containing 1% w/v DTT for 15 min, followed by a second incubation in 2.5% w/v iodoacetamide in the same equilibration solution for 15 min. 2-DE was performed using 0.7 cm thick, 18 × 18 cm linear gradient gels (7.5-17.5%) in a Protein ∏ xi 2-D Cell apparatus (Bio-Rad). The gels were stained with silver or Coomassie Brilliant Blue G250 to visualize proteins. Images of stained gels were digitized with a densitometer (Versa Doc Imagin System 1000™, Bio-rad). The density of spots was detected and counted by both automation and manual spot-detection, and statistically analyzed with PDQuest software (Version 7.1.1, Bio-Rad). Protein expression data from gels were normalized for the total density presented in gel images. Protein identification procedure was described in detail in Additional file 2.
Western blot analysis
Western blot analysis was performed as described previously [3, 11]. Molecular weight standards were obtained from New England Biolabs (Ipswich, MA, USA). Membranes were probed with primary antibodies recognizing the following proteins: SOD-Cu/Zn, SOD-Mn, and aldose reductase were gifts from Dr. Seo H-G (Gyeongsang National University, Jinju, South Korea). peroxiredoxin-2, -4, Bax and Annexin-A5, HSP-27 antibodies were purchased from Santa Cruz (CA, USA). Active- and pro-caspase-3, active- and pro-caspase-8, active PARP was from MERK (Darmstadt, Germany). Bcl-2 and actin antibody were from Abcam (Cambridge, UK) and CHEMICON (Temecula, CA, USA), respectively. Thereafter, the membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) and subjected to enhanced chemiluminescence analysis (Amersham, Piscataway, NJ, USA). An anti-actin antibody (1:500, CHEMICON International) was used to verify equal protein loading. Signals were visualized by using the ECL kit (Amersham). Band intensities of each protein expression were quantified by Image processing and analysis using Image J 1.23 (NIH image).
TdT-mediated dUTP-X Nicked End labeling (TUNEL) assay
TUNEL assays were performed as described previously . Briefly, tissue sections were incubated with TUNEL mix (0.3 U/μL calf thymus terminal deoxynucleotidyl transferase, 7 pmol/μL biotin dUTP, 1 mM cobalt chloride in 1 × reaction buffer in distilled water) and then washed. Sections were saturated and then treated with a 1:20 dilution of ExtraAvidin peroxidase antibody. After washing, sections were incubated in DAB staining solution [1.24 mg DAB, 25 μL 3% NiCl2, 152 μL 1 M Tris-HCl (pH 7.5) in 2 ml distilled water]. Slides were mounted in crystal mount (Biomeda, Foster City, CA, USA) and visualized by microscopy.
All experimental data represent the means ± standard deviation (SD). Analysis of angiogenesis-related gene expression pattern in figure two were performed three times by using 10 scNT-N and 6 scNT-MUC piglets and the statistical significance was confirmed by using t-test. Wound healing and tubule formation in figures five and six were analyzed by using 2 controls, 3 scNT-N and 6 scNT-MUC samples with three replicates and the statistical significance was calculated by ANOVA and confirmed by Duncan's multiple range procedure. Protein and mRNA expression levels (figure seven) were examined in 3 controls, 6 scNT-MUC with three replicates and the statistical significance was confirmed by t-test.
Results and discussion
High incidence MUC in scNT-derived piglet clones
Efficacy of scNT-derived piglet production
118.4 ± 2.408
114.5 ± 0.7
When we examined the expression of PECAM-1 (CD31), which is abundantly expressed in ECs , scNT-MUC expressed significantly lower levels of PECAM-1 than control and scNT-N (Figure 1D). PECAM-1 is an efficient signaling molecule that functions in diverse aspects of vascular biology, including angiogenesis, platelet function and the regulation of leukocyte migration . Thus, impaired expression of PECAM-1 in the umbilical cord could be a contributing factor in the development of umbilical cord malformation.
Umbilical-derived vein endothelial cells of scNT-MUC clones exhibit abnormal tubular junctions and tubule formation
scNT umbilical cords express low levels of glycolytic- and cell motility-associated proteins
We performed real-time RT-PCR to validate some of the data derived from 2-DE analysis, and to investigate the relationship between the mRNA and protein expression levels of cytoskeletal- and motility-related proteins. As shown in Figure 7B, the mRNA levels of destrin, WD-repeat protein 1 (WD-1), desmin, ubiquitous tropomodulin 3 (TMOD3), cofilin, transgelin, tropomyosin 1 alpha (TPM-1), calpain small subunit (CAPNS1), tropomyosin 1 beta (TPM-2), actin and tropomyosin alpha 4 chain (TPM-4) were significantly lower in scNT-MUC (p < 0.05) than in control. In particular, there was a pronounced difference in the mRNA expression level of destrin (Figure 7B, p < 0.01). When we directly compared protein and mRNA expression patterns, with the exception of alpha-centractin, all cytoskeletal and motility-related proteins exhibited similar patterns of down-regulation at both the protein and mRNA levels in scNT-MUC (Figure 7B). Thus, mRNA expression levels in the umbilical cord closely mirrored protein expression levels. The exception was alpha-centractin, which was up-regulated at the protein level, and down-regulated at the mRNA level in scNT-MUC. These results suggested that alterations in the expression of proteins involved in the cytoskeleton and motility lead to defects in placental EC migration and tubule formation. Defects in EC signal transduction and function could lead to the characteristic disorganization of myofibers in scNT-MUC-derived placentas.
Up-regulation of apoptotic proteins and down-regulation of oxidative repair proteins in scNT-MUC
A number of proteins involved in apoptosis and cell cycle signaling were consistently altered in scNT-MUC relative to control umbilical cords (see Additional file 4). In particular, proteins in two major categories of apoptosis-related proteins, lipid-binding and oxireductase activity, were frequently altered in scNT-MUC. The lipid-binding apoptosis-related proteins Annexin A1, A2, and A5 were up-regulated in scNT-MUC. Annexins are structural proteins that bind to phospholipids in a Ca+2-dependent manner, and are well-characterized apoptosis biomarkers . The expression levels of lamin A and HSP27 were also up-regulated in scNT-MUC as compared to controls. Lamin is cleaved by members of the interleukin-converting enzyme family during apoptosis , and HSP27 (and HSP71) induces apoptosis through the activation of the caspase cascade . These results were indicative of increased levels of apoptosis in scNT-MUC. The oxireductase activity-related proteins peroxiredoxin (Prx)-2 and -4 and Cu/Zn superoxide dismutase (SOD) were significantly down-regulated in scNT-MUC, which suggested that damaging reactive oxygen species (ROS) accumulate in scNT-MUC and contribute to apoptosis .
Given the apparent increase in the expression of apoptosis-related proteins in scNT-MUC, we next examined the expression of several proteins that are key factors in programmed cell death. The level of expression of the anti-apoptotic protein Bcl-2 was lower in scNT-MUC than scNT-N, whereas Bax, active poly (ADP-ribose) polymerase (PARP) and pro/active caspases 3 and 8 were significantly up-regulated (Figure 8C and 8D). PARP is an important substrate of caspase-3 that is cleaved from a 112-kDa fragment into an 85-kDa fragment upon caspase-3 activation [31, 32, 34]. These results provided additional evidence of active apoptosis in scNT-MUC. While the elimination of unwanted cells during development is essential, inappropriate or elevated apoptosis in the umbilical cord can result in destruction or weakening of the tissue. The umbilical cord is essential during development, as it provides nutritional, endocrine and immune support to the fetus. When these functions are compromised due to elevated apoptosis, low placental weights and low birth weights can ensue.
TUNEL analysis of organs from scNT-MUC piglets
Recent studies from our laboratory suggest that the low success rate of scNT cloning is due to placental abnormalities rather than to cumulative genomic damage [3, 7, 11, 12]. The data from the current study are consistent with this hypothesis, and indicate that small placentas derived from scNT-MUC inflict chronic pressure on the developing fetus, resulting in compromised organ development. Thus, MUC contribute to placental insufficiency, and ultimately influence fetal development, malformation and birth rates.
Our observations suggest that due to MUC, the blood flow may be reduced between placenta and fetus, resulting in an increase in apoptosis in the umbilical cord. Because the umbilical cord plays a fundamental role in transporting metabolites between mother and fetus, MUC also contribute to placental insufficiency by preventing the removal of harmful materials from fetal circulation. MUC might also promote hypoxia and the accumulation of CO2, while diminishing O2 levels in fetal organs, stimulating apoptosis in the developing fetus. Thus, the functional consequences of MUC in scNT-derived animals are potentially severe, and include not only placental insufficiency, fetal abnormalities and mortality, but also fetal malformations, preterm birth, and low birth weight. Our data suggest that these effects are due to specific molecular defects that lead to the development of HUA and/or placental insufficiency. In summary, the results of the current study provide several clues to a better understanding of the molecular mechanisms of angiogenesis during umbilical cord development, as well as a robust model for the study of HUA syndrome in humans.
List of abbreviations
calpain small subunit
2-dimensional gel electrophoresis
hypoplastic umbilical arteries
inner longitudinal smooth muscle
malformed umbilical cords
outer circular smooth muscle
poly (ADP-ribose) polymerase
platelet endothelial cell adhesion molecule-1
reactive oxygen species
porcine umbilical vein-derived endothelial cells
somatic cell nuclear transfer
single umbilical artery
tropomyosin 1 alpha
tropomyosin 1 beta
tropomyosin alpha 4 chain
TdT-mediated dUTP-X Nicked End labeling
vascular endothelial growth factor
WD-repeat protein 1
This study was supported in part by the Research Project of Biogreen 21 (20070401-034-033) from RDA, Republic of Korea.
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