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

Abstract

Abstract. Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P < 0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

Functional consequences of mitochondrial mismatch in reconstituted embryos and offspring

“…The influence of potential nuclear-cytoplasmic incompatibility post SCNT is largely unknown. Solubilized hepatic mitochondrial proteins from SCNT pigs produced via the microinjection of M fibroblasts into L oocytes were comprehensively analyzed by fluorescent labeled two-dimensional difference gel electrophoresis (2-D DIGE) [17]. The 2-D DIGE analysis revealed the differential expression of three proteins among the M and L breeds, all of which showed high expression in M pigs.…”

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

“…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]. Although alteration of liver mitochondrial protein expression levels was observed in adult SCNT pigs, it was not reflective of breed differences associated with recipient oocytes.…”

“…Up-regulation of proteins controlling glucose metabolism and ROS suggests that the accumulation of ROS may induce apoptosis in the pancreas in adult SCNT pigs [78]. Down-regulation of the aldehyde dehydrogenase family 4 member A1, which acts as a negative regulator of p53-dependent apoptosis [88], was found in liver mitochondria derived from adult SCNT pigs [80]. Similarly, an up-regulated 78 kDa glucose-regulated protein precursor, which enhances ER (endoplasmic reticulum) stress associated with cell death [89], was observed in liver mitochondria of adult SCNT cattle [82].…”