29 PROTEOMIC ANALYSIS OF CLONED TERM PLACENTA DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER CATS

Abstract

The normal placenta formation and development affect subsequent survival and development of fetus. Somatic cell nuclear transfer (SCNT) is still associated with functional and structural abnormalities of placentation in cloned pregnancy. Moreover, the molecular basis of placenta derived from cloned embryos is poorly understood. To determine the abnormal expression of proteins of cloned term placenta (CTP; n = 3) compared with control placenta (n = 3), a proteomic analysis was performed using 2-DE, MALDI-TOF MS, and MALDI-TOF/TOF MS/MS. The cloned kittens were killed 1 month after Caesarean section. The results showed that 42 proteins were significantly up/down expressed in the CTP (P < 0.05). In CTP, thirty proteins were up-regulated, such as apoptosis-related cathepsin D (CD), annexin A1, and heat shock protein 27, and 12 proteins were down-regulated, such as prohibitin (PHB). Abnormal expression of CD and PHB may be related to induced reactive oxygen species (ROS) resulting in promoted cellular senescence and apoptosis. The expression patterns of CD and PHB were validated by Western blotting and immunofluorescence assay. Additionally, we also confirmed the abnormal expression of SOD (up-regulated) and CAT (down-regulated) enzymes in the CTP. Increased generation of ROS in the CTP was related to decreased mitochondrial membrane potential as detected in placenta tissues by MitoTracker green FM (MTG; 1 μM, Invitrogen, Carlsbad, CA). ROS-induced mitochondrial damage accelerated telomere loss [as detected by IQ-fluorescent in situ hybridization (FISH)] compared with control. ROS is an important modulator of telomere loss and that telomere-driven cellular senescence is primarily a stress responsive. Thus, the data suggest that combined abnormal proteins expression were associated with hindered development of cloned placenta and viability of fetus. Therefore, if we can solve this problem, the efficiency of SCNT and transgenic cat production will be increased, which in turn, may be applied as a potential means of restoration of endangered animals.