Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death

Abstract

BackgroundSomatic 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.ResultsMicroscopic 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.ConclusionThese 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.

Comparative Proteomic Analysis of Liver Mitochondrial Proteins Derived from Cloned Adult Pigs Reconstructed with Meishan Pig Fibroblast Cells and European Pig Enucleated Oocytes

“…Lee et al [18] assessed the global protein expression profiles in term placentas from SCNT pigs and found that the proteins closely involved in the apoptotic signaling pathway were different in comparison with controls. In malformed umbilical cords of SCNT piglets, proteins prevention of oxidative stress were downregulated, while molecules involved in apoptosis were upregulated [20]. In the present study, two novel proteins including HMG-CoA synthase were identified as common differences in the SCNT pigs.…”

“…Accordingly, many abnormalities observed in SCNT fetuses and offspring may be influenced by deficiencies in mitochondrial function. Recently, comparative proteomic analyses of SCNT piglets and placentae from SCNT pigs were reported [18][19][20]35]. Two-dimensional gel electrophoresis analysis revealed changes in the responses of several detoxification-related proteins to stress and inflammation in sudden death observed in SCNT piglets with extensive hepatopneumonic apoptosis [17].…”

“…Recently, global comparative proteomic analysis was performed to identify the proteins that cause problems in animal cloning [17][18][19][20]. Different expression levels in disease-relevant proteins and/or in apoptosis were observed in piglets, fetuses and placentae of offspring derived from SCNT pigs [17][18][19][20].…”

Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs

“…MtDNA genotypes were determined by PCR-mediated single-strand conformation polymorphism analysis using the variable region of D-loop [6,7]. CA3 and CA4 exhibited a mixture of genotypes with D-mtDNA [79] more sequence variants in a cell, a genetic bottleneck in the female germ line will generally favor the transmission of a single mitochondrial population (homoplasmy) [13,27,28]. However, in SCNT animals both stable and unstable transmission of D-mtDNA was found in porcine and bovine G n s [10,17].…”

“…Two-dimensional differential gel electrophoresis (2D DIGE) is becoming more popular in protein quantification with the inclusion of an internal standard, providing a technology that greatly reduces experimental variation [74,75]. Recently, differentially expressed proteins of SCNT piglets and placentae from SCNT pigs and cattle were reported using comparative proteomic analyses [76][77][78][79]. We investigated the differences of protein expression profiles of mitochondria in adult SCNT pigs derived from Chinese pig (Meishan breed) fibroblasts and European pig enucleated oocytes (maternal Landrace breed) [17,29] using a 2D DIGE system [80].…”