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Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.

Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Here, we performed protein profiles in preterm- and term-derived human umbilical cord by using 2DE. Approximately 200 different proteins were identified between preterm- and term-delivered umbilical cords. Among them, 48 proteins were identified. A comparison of preterm proteome to that of term proteome revealed potential candidates for biomarkers, such as hypoxia-inducible proteins, phosphorylated heat-shock protein 27 (HSP27), transgelin, vimentin, and transferrin that are specific to preterm umbilical cords. Especially, HSP27 in preterm-derived umbilical cords shows a significant increase in the mono- and tetra-phosphorylation. The real importance of all of HSP27 phosphorylation as well as hypoxia-inducible factor 1alpha, and glyceraldehyde 3-phosphate dehydrogenase require further validation in vitro and in vivo; nevertheless, we believe that they could represent promising diagnostic targets for detection of sudden early delivery. In conclusion, the results of the current study may provide important insights into the molecular mechanisms underlying umbilical cord development.

In this study, we examined whether Hanganutziu-Deicher (H-D) antigens are important as an immunogenic non-α1,3-galactose (Gal) epitope in pigs with a disrupted α1,3-galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The α1,3-galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote α1,3-galactosyltransferase gene knockout (GalT-KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of α1,3-galactosyltransferase activity when compared to those of control. Enzyme-linked lectinosorbent assay showed that the heterozygote GalT-KO pig had more sialylα2,6- and sialylα2,3-linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT-KO pig had a higher N-glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT-KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P

Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5'-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.

Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) andα2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level ofα-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production ofN-glycolylneuraminic acid (Neu5Gc) due to an increase ofα2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.

In this investigation, 22 cloned male piglets were obtained by male fetal fibroblast-cell-derived nuclear transfer. Eighteen of the cloned animals died. The two cell lines did not differ significantly with regard to efficiency of live piglet production. The gross anatomy of the testes of male piglets that died was normal. However, one piglet displayed Leydig cell hypoplasia (LCH). No anatomical defects were detected in the testes of other cloned male piglets. TUNEL analysis of the testis with LCH revealed significant apoptosis in the Leydig cells, while apoptosis was rarely detected in Sertoli cells and spermatogonia. In contrast, testes from the remaining 17 piglets that died appeared normal in size, and their Sertoli and Leydig cell numbers were comparable to those in control piglet testes. Although cloned piglets were derived from fibroblasts obtained from the same fetus, phenotypic instability between cells used for the production of somatic cell cloned piglets suggests that abnormalities in male cloned piglets are caused not by technical problems and/or reprogramming effects, but rather by epigenetically and/or genetically damaged cell-specific effects.

The regulatory mechanisms of glycosyltransferase activities are quite complicated and are still poorly understood. Thus far, several reports have appeared concerning the nature of regulatory proteins for intracellular glycosyltransferase activities. Those include a specific molecular chaperon for core 1 b3-galactosyltransferase, a testis-specific inhibitor for N-acetylglucosaminyltransferase (GnT)-I, and a cellular factor for dictating the functional localization of GnT-III in the intra-Golgi sub-compartment. Quite recently, an additional novel regulatory protein for glycosyltransferase activity was unequivocally identified based on purifying, identifying, and characterizing ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) 3 as an intrinsic inhibitory factor for GnT-IX(Vb) in murine neuroblastoma Neuro2a cells. The enzyme was found to inhibit GnT-IX via ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate UDP-GlcNAc, with the resulting generation of UMP, a potent competitive inhibitor of GnT-IX. Such a novel regulatory mechanism of glycosyltransferase activity would be responsible for modulating the total cellular glycosylation profile and various cellular functions.

Glycosides, 1-O-benzyl-α-glucoside (BG) and 1- O-benzyl-α-maltoside (BM), were synthesized from soluble starch and benzyl alcohol by transglycosylation with an α-amylase in a water system. BG was mostly obtained in a reaction mixture of pH 5.0, while BM was synthesized in pH 8.0. The synthesized glycosides had α-configuration linkage between sugar and benzyl alcohol. The BG was rapidly hydrolyzed to benzyl alcohol and glucose by α-glucosidase. The BM was hydrolyzed to BG and glucose below pH 5.0 by the α-amylase used for its synthesis but it was not hydrolyzed above pH 8.0.

Determining glycosyltransferase activities gives a clue for better understanding an underlying mechanism for glycomic alterations of carrier molecules. N-glycan branch formation is concertedly regulated by cooperative and competitive activities of N-acetylglucosaminyltransferases (GnTs). Here, we describe methods for large scale preparation of the oligosaccharide acceptor substrate, fluorescence-labeling of oligosaccharides by pyridylamination, quality control, and reversed phase HPLC-based measurement of GnT activities including GnT-III, IV, V, and IX.

Determining glycosyltransferase activities gives a clue for better understanding an underlying mechanism for glycomic alterations of carrier molecules. N-glycan branch formation is concertedly regulated by cooperative and competitive activities of N-acetylglucosaminyltransferases (GnTs). Here, we describe methods for large scale preparation of the oligosaccharide acceptor substrate, fluorescence-labeling of oligosaccharides by pyridylamination, quality control, and reversed phase HPLC-based measurement of GnT activities including GnT-III, IV, V, and IX.

Synthesis of a bioemulsifier using a lipase from Pseudomonas sp. with fructose and vinyl laurate was carried out in anhydrous pyridine. The synthetic product was identified as laurylfructose with an emulsifying activity on various hydrocarbons, edible oils and petroleum oils. The compound reduced the surface tension of distilled water from 72 mN/m to 29 mN/m and the interfacial tension of water/n-hexadecane from 50 mN/m to 6 mN/m.

Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Here, we performed protein profiles in preterm- and term-derived human umbilical cord by using 2DE. Approximately 200 different proteins were identified between preterm- and term-delivered umbilical cords. Among them, 48 proteins were identified. A comparison of preterm proteome to that of term proteome revealed potential candidates for biomarkers, such as hypoxia-inducible proteins, phosphorylated heat-shock protein 27 (HSP27), transgelin, vimentin, and transferrin that are specific to preterm umbilical cords. Especially, HSP27 in preterm-derived umbilical cords shows a significant increase in the mono- and tetra-phosphorylation. The real importance of all of HSP27 phosphorylation as well as hypoxia-inducible factor 1alpha, and glyceraldehyde 3-phosphate dehydrogenase require further validation in vitro and in vivo; nevertheless, we believe that they could represent promising diagnostic targets for detection of sudden early delivery. In conclusion, the results of the current study may provide important insights into the molecular mechanisms underlying umbilical cord development.

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs.

Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5'-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.

Somatic cell nuclear transfer (scNT)-derived pig placenta tissues of gestational day 30 displayed avascularization and hypovascularization. Most of the cytotrophoblast-like cells of the developing scNT-derived placenta villi were improperly localized or exhibited impaired migration to their targeting loci. Id-2, Met, MMP-9, and MCM-7 were barely detectable in the cytotrophoblast cells of the scNT-derived placenta villi. Active MMP-2 and MMP-9 expression was significantly down-regulated in the scNT-embryo transferred recipient uteri. scNT clones exhibited a hypermethylated pattern within the pig MMP-9 promoter region and the significance of GC box in the regulation of MMP-9 promoter activity. Marked apoptosis was observed in the developing endometrial gland of scNT-embryo transferred recipient uteri. Collectively, our data strongly indicated that early gestational death of scNT clones is caused, at least in part, by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness due to the down-regulation of active MMP-9 expression. Developmental Dynamics 240:627–639, 2011. © 2011 Wiley-Liss, Inc.

Previously, we have successfully produced nine cloned piglets using Duroc donor cells. Among these clones, one showed distinct depigmentation of the skin and hair color during puberty. In this study, we selected a clone with depigmentation to investigate the etiology of the anomaly in somatic cell nuclear transfer. We hypothesized that genes related to Waardenburg syndrome (Mitf, Pax-3, Sox-10, Slug, and Kit) are closely associated with the depigmentation of pig, which was derived from somatic cell nuclear transfer (scNT). Total RNA was extracted from the ear tissue of affected and unaffected scNT-derived pigs, and the transcripts encoding Mitf, Pax-3, Sox-10, and Slug, together with the Kit gene, were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed. The cDNA sequences from the scNT pig that showed progressive depigmentation did not reveal a mutation in these genes. Although we did not find any mutations in these genes, expression of the genes implicated in Waardenburg syndrome was severely down-regulated in the affected scNT pig when compared with unaffected scNT pigs. This down-regulation of gene expression may result in a previously undescribed phenotype that shows melanocyte instability, leading to progressive loss of pigmentation. Developmental Dynamics 238:1701–1708, 2009. © 2009 Wiley-Liss, Inc.

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.

In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, five piglets were stillborn, 22 scNT piglets died suddenly within the first week of life, and 1 piglet died after 40 days. Twelve scNT piglets are still healthy. The birth weights of compromised scNT piglets in comparison with those of normal scNT piglets are significantly reduced (0.80 +/- 0.29 vs 1.27 +/- 0.30 kg, p0.05), in spite of longer gestation (114 versus 120 day). Significant findings from histological examinations showed that approximately 25% (7/28) of scNT piglets showed severe congestion of lung and liver or neutrophilic inflammation in brain indicating that unexpected phenotypes can appear as a result of somatic cell cloning. Two-dimensional gel electrophoresis experiments revealed changes in the responses of several detoxification-related proteins related to stress and inflammation and found significant alterations in myocardium-specific proteins, indicating hemodynamic disorder. scNT piglets that survived to adulthood did not show any abnormality except skin and hair color depigmentation. The present study suggests that cerebromeningitis and hemodynamic disorder are a major risk factor for sudden early death of scNT piglets. Although we cannot completely exclude the possibility that scNT piglets are susceptible to specific respiratory infections, our data suggests that the early death of scNT clones is due to cardiopulmonary functional abnormalities and cerebromeningitis.

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin alpha and beta are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and alpha1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin alpha derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin alpha. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use.

Background 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. Results 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. Conclusion 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.