High incidence MUC in scNT-derived piglet clones
In this study, we carried out scNT using each donor fibroblast cell derived from Duroc, Berkshire and 3 way hybrids (Landrace × Duroc × Yorkshire). scNT embryos were transferred and successfully produced 65 cloned piglets from 24 surrogates (Table ). The number of offsprings was variable depending on the surrogates ranging from 1 to 6 piglets. Genetic identification for scNT clones was confirmed by using porcine DNA microsatellite markers (SWR1120, SW1311, and SW1327) (data not shown). Of these 9 piglets were derived from Duroc, 12 piglets from 3 way hybrid, and 44 piglets from Berkshire: among them, 14 piglets (21.5%) were stillborn, 38 scNT piglets (58.5%) died suddenly within the first week of life, and 3 piglets (4.6%) died during puberty. To date, only 10 scNT pigs (4 piglets were derived from Duroc, 3 piglets from 3 way hybrid, and 3 clones from Birkshire) are still alive and healthy. Analysis of 65 term scNT-derived piglet clones revealed that nine had severe MUC, a prevalence of 13.9%. Of the 56 scNT piglets with normal umbilical cords, twelve (21.4%) were stillborn, 33 (58.9%) died suddenly within the first week of life, and one (1.8%) died during puberty. Ten (17.9%) of the 56 scNT-N pigs survived into adulthood, and remain alive and healthy. Of the nine scNT piglets with MUC, two (22.2%) were stillborn and four died within the first week of life. Of the remaining scNT-MUC piglets, two reached puberty but then died between 8-12 months of age. Only one of the nine scNT-MUC piglet clones survived to adulthood. The birth weight (0.882 ± 0.243 kg; n = 9) and placenta weight (0.24 ± 0.04 kg; n = 9) of scNT-MUC clones were significantly lower (p < 0.05) than those of scNT-N clones (1.390 ± 0.675 kg and 0.28 ± 0.05 kg, respectively; n = 10) and control piglets [1.352 ± 0.112 kg (n = 20) and 0.33 ± 0.08 kg (n = 35), respectively]. Further analyses were carried out by using Berkshire-derived scNT clones and controls to minimize breeder variability.
Efficacy of scNT-derived piglet production
Upon histological examination, scNT-MUC exhibited severe tissue damage as compared to control and scNT-N, including tissue swelling (Figure , arrows). Microscopic analysis revealed complete occlusive thrombi in the veins and arteries of scNT-MUC, but not in control and scNT-N (Figure ). scNT-MUC also exhibited areas of irregular outer circular smooth muscle (OCSM) and unclear boundaries in the inner longitudinal smooth muscle (ILSM) (Figure ). Analysis of umbilical cords using the TUNEL assay to detect apoptotic nuclei clearly showed that there was a high level of apoptosis in the ILSM of scNT-MUC, whereas apoptotic cells were rarely detected in the ILSM of scNT-N umbilical cords (Figure ; bottom panel). These results suggested that there is severe vessel damage to both veins and arteries in scNT-MUC.
Figure 1 Morphological and histological analysis of control, scNT-N and scNT-MUC term piglets. A) scNT-MUC exhibited more swelling and severe damage as compared to control and scNT-N. Of particular interest, the tissues of scNT-MUC were extremely fragile as compared (more ...)
When we examined the expression of PECAM-1 (CD31), which is abundantly expressed in ECs [22
], scNT-MUC expressed significantly lower levels of PECAM-1 than control and scNT-N (Figure ). 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 [23
]. Thus, impaired expression of PECAM-1 in the umbilical cord could be a contributing factor in the development of umbilical cord malformation.
EC activation is initiated by the binding of proangiogenic factors, such as vascular endothelial growth factor (VEGF), to their cognate receptors on endothelial cells, an event that induces the activation of angiogenic signaling pathways [24
]. VEGF is one of the most potent inducers of vascular permeability, and specifically targets ECs through binding to specific endothelial cell surface receptors, including VEGFR-1 and VEGFR-2. The activation of VEGFR-1 induces endothelial permeability [25
], while VEGFR-2, which is expressed exclusively in ECs, appears to play a pivotal role in EC differentiation and vasculogenesis [26
]. Angiopoietin-1 (Ang-1), a key molecule in the regulation of embryonic vascular development, binds to and activates the endothelial specific receptor Tie2, while Ang-2 is required for subsequent postnatal vascular remodeling [27
]. scNT-MUC had significantly lower mRNA expression levels of VEGF, Ang-1, Ang-2, VEGFR-1 and Tie-2 (Figure ), as compared to scNT-N umbilical cords. The expression of VEGFR-2 was also lower in scNT-MUC than scNT-N umbilical cords. These results suggested that ECs in scNT-MUC fail to elicit mitogenic responses and/or form tubules due to the down-regulation of vascular signaling molecules (i.e. VEGF, angiopoietin and/or their cognate receptors) that play essential roles in vascular development. These defects in EC signaling might be responsible, at least in part, for the developmental abnormalities in scNT-MUC.
Figure 2 Angiogenesis-related gene expression in scNT-N and scNT-MUC. Real-time RT-PCR was carried out using primers that were specific for VEGF, Ang-1 and Ang-2, and their cognate receptors VEGFR-1, VEGFR-2 and Tie-2, respectively. Real-time RT-PCR was independently (more ...)
We also observed several abnormalities in scNT-MUC-derived placentas, including villous hypovascularity and cytotrophoblast and syncytotrophoblast hypoplasia (Figure ). Placentas derived from scNT-N exhibited similar abnormalities (Figure , middle column), however, there were some subtle differences between scNT-MUC- and scNT-N-derived placentas (Figure , right column). Most scNT-MUC-derived placentas had fewer blood vessels than normal placentas (data not shown), and exhibited severe aponecrosis of the cytotrophoblast and syncytotrophoblast cells in the developing villi (Figure , lower right column). In placentas in which these cells were intact, there was a significant decrease in the expression of the cytotrophoblast biomarker MCM-7 (Figure , bottom section). These observations suggested that the placenta abnormalities in the scNT-MUC clones may be associated with a functional abnormality of the ECs and that this may affect the size and survival of the developing fetus.
Figure 3 Photomicrographs of cross-sections of control, scNT-N- and scNT-MUC-derived term placentas. Dewaxed paraffin sections were stained with hematoxylin and eosin. The villi and column trophoblast cells developed normally in control term placentas, but were (more ...)
Umbilical-derived vein endothelial cells of scNT-MUC clones exhibit abnormal tubular junctions and tubule formation
PUVECs were obtained from the veins of control, scNT-N and scNT-MUC, as described previously [18
]. We established five cell lines from nine scNT-MUC piglets, and determined the purity of each PUVEC line by immunostaining using anti-PECAM-1 (CD31) or anti-Factor Vlll (VWF) antibodies (data not shown). As shown in Figure , most of the cells derived from control, scNT-N and scNT-MUC piglets were positive for CD31, which indicated that they are umbilical-derived EC lines of relatively high purity.
Figure 4 Immunohistochemical analysis of ECs derived from control (a), scNT-N (b), and scNT-MUC (c). Cells were cultured in vitro and then stained using an anti-CD31 (PECAM-1, red) antibody, followed by counterstaining of nuclear DNA with DAPI (blue). Insets show (more ...)
To determine whether the changes in angiogenesis-related gene expression that we observed earlier in scNT-MUC affected EC motility, we performed a migration assay using PUVEC cell lines from control, scNT-N and scNT-MUC piglets. PUVECs were grown to confluence and an area of the dish (the so-called wound) was scraped clear of cells by applying suction with a narrow tip (Figure , and --). Cells were allowed to incubate for 24 hr, and then examined by microscopy. The migration of scNT-MUC-derived PUVECs was significantly impaired as compared to control and scNT-N-derived PUVECs (Figure , and ), with statistically significant differences in the numbers of migrating and proliferating cells (Figure ).
Figure 5 Wound healing assay. scNT-MUC-derived PUVECs (e and f) had significantly lower levels of cell migration than control (a and b) and scNT-N (c and d) PUVECs. The graph shows the number of cells that migrated into the wound 24 hr after wounding. The data (more ...)
In addition to the migration and proliferation of ECs, proper vascularization requires vascular maturation, during which the rate of vascular sprouting is attenuated to prevent vascular collapse. Vascular maturation involves the recruitment of both perivascular and smooth muscle cells [28
]. We next examined the ability of scNT-MUC-derived PUVECs to form EC junctions and tubules. Cells were plated on Matrigel, and allowed to incubate for 6 hr. Control and scNT-N PUVECs formed networks consisting of cellular aggregates (nodes), with branches of elongated strands of cells (Figure and ). scNT-MUC-derived PUVECs also formed EC junctions, but the networks consisted of a small population of nodal cells, and tubular branches with an abnormal morphology (Figure ). scNT-MUC-derived PUVECs also formed significantly shorter tubules than control and scNT-N PUVECs (Figure ). These results suggested that decreased expression of angiogenesis-related genes in scNT-MUC results in defective PUVEC migration and tubule formation.
Figure 6 In vitro junction and tubule formation assay. Control and scNT-N-derived PUVECs formed an extensive capillary-like network 24 hr after plating onto Matrigel (a and b), while scNT-MUC PUVECs formed a truncated and abnormal network (c). Cells were viewed (more ...)
scNT umbilical cords express low levels of glycolytic- and cell motility-associated proteins
The umbilical cords of rodents and large mammals, including humans, are glucose-dependent tissues that undergo limited mitochondrial respiration, and rely predominantly on the anaerobic conversion of glucose to lactate [29
]. We compared the proteomes of scNT-MUC and control umbilical cords by 2-dimensional gel electrophoresis (2-DE) (see Additional file 3
), and found that the expression levels of several glycolytic enzymes were lower in scNT-MUC (Figure ). For example, phosphoglucomutase-like protein 5, triosephosphate isomerase, phosphoglycerate kinase 1 and L-lactate dehydrogenase B chain were down-regulated by 2.4-, 7.5-, 1.3-, and 1.4-fold, respectively. By comparison, a number of enzymes involved in aerobic metabolism (TCA cycle) were up-regulated in scNT-MUC (Figure and see Additional file 4
). These results suggested that, unlike normal umbilical cords, scNT-MUC lack sufficient protection from oxidative damage due to the decreased expression of key glycolytic enzymes and antioxidant proteins. Several other classes of protein whose expression differed between control umbilical cords and scNT-MUC were identified, and included proteins involved in detoxification, chaperone activity, signal transduction and cytokine activity (see Additional file 4
). Of note, several proteins involved in the regulation of the cytoskeleton and motility were differentially expressed in control umbilical cords and scNT-MUC.
Figure 7 Proteomic analysis of control and scNT-MUC. A) 2-DE analysis of differentially expressed proteins in control and scNT-MUC. Several glycolytic enzymes and TCA-related enzymes were down- and up-regulated, respectively, in scNT-MUC relative to control. B) (more ...)
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 , 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 , 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 ). 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 [30
]. 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 [31
], and HSP27 (and HSP71) induces apoptosis through the activation of the caspase cascade [32
]. 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 [33
To identify the minimal repertoire of molecules involved in the development of MUC while also controlling for variation due to scNT, we carried out a comparative proteomic analysis of scNT-N and scNT-MUC using 2-DE followed by MALDI-TOF/TOF mass spectrometry. We identified 70 protein species that appeared to be differentially expressed in scNT and scNT-MUC (see Additional file 5
). Among them, 35 were present at higher levels in scNT-MUC, while 25 were present at lower levels as compared to scNT-N. Additional file 6
summarizes the main properties of 40 of the proteins identified that were differentially regulated in scNT-MUC and scNT-N. The expression of apoptosis-related proteins (annexin A2, A5, and HSP 27) was up-regulated, whereas the expression of cell motility and structural proteins (heat-shock 20 kDa like-protein p20, transgelin, tubulin-specific chaperone A, LIM and SH3 protein 1, destrin), and detoxification proteins [Prx-2, -4, SOD-Cu/Zn, aldose reductase (AR)] was down-regulated (Figure and ). Western blot analysis confirmed the up-regulation of annexin A5 and HSP27, and the down-regulation of AR, SOD-Cu/Zn, SOD-Mn, Prx-2 and Prx-4 in scNT-MUC (Figure and ).
Figure 8 Proteomic analysis of scNT-N and scNT-MUC by 2-DE and Western blot. A) Representative up- and down-regulated proteins from scNT-N and scNT-MUC are shown. B and C) Western blot analysis of ROS- and apoptosis-related proteins in scNT-N and scNT-MUC. The (more ...)
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 and ). 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
]. 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
To investigate the relationship between MUC and fetal malformation, we examined several postnatal fetal organs by microscopy (Figure ). We observed severe congestion in the lung and liver of scNT-MUC piglets (Figure and ). The affected tissues had a prominent red color due to engorgement with oxygenated blood and exhibited severe peliosis. There was also evidence of abnormal stromal cell proliferation and tubular degeneration in the lung and liver (Figure and , arrows). Of note, distinct areas of calcification were detected in the placenta and kidney (Figure and , arrowheads). We also measured the level of apoptosis in control and scNT-MUC-derived placenta, lung, liver and kidney using the TUNEL assay (Figure ). Whereas apoptotic cells were barely detectable in control placentas (Figure ), there was a marked elevation in apoptosis in organs derived from scNT-MUC clones (Figure ). Apoptosis in scNT-MUC placentas was restricted primarily to cytotrophoblast and syncytotrophoblast cells, with the exception of a small sub-population of cells attached to the basal membrane of the villi (Figure and Figure , lower right panel).
Figure 9 Analysis of organs derived from control and scNT-MUC piglets. A) Microscopic analysis revealed evidence of severe calcification in scNT-MUC-derived placenta (e) and kidney (h). Lung (f) and liver (g) sections derived from scNT clones with MUC showed abnormal (more ...)
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
]. 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.