Mitochondrial dysfunction influences apoptosis and autophagy in porcine parthenotes developing in vitro.
Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial-mediated apoptosis is the production of reactive oxygen species (ROS), which include hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrite. Recently, several studies have indicated that ROS may also be involved in the induction of autophagy. In the present study, we used H(2)O(2) to induce mitochondrial stress, examined apoptotic- and autophagic-related gene expression and observed LC3 protein (autophagosome presence marker) expression in porcine parthenotes developing in vitro. In porcine four-cell parthenotes cultured for 5 days in NCSU37 medium containing 0.4% BSA, the developmental rate and mitochondrial distribution did not differ from that of the group supplemented with 100 µM H(2)O(2) but was significantly decreased in the group supplemented with 500 µM H(2)O(2) (P<0.05). Transmission electron microscopy (TEM) indicated that whereas normal shaped mitochondria were observed in blastocysts from the control group, abnormal mitochondria (mitophagy) and autophagic vacuoles were observed in blastocysts from the group that received 500 µM H(2)O(2). Furthermore, addition of H(2)O(2) (100 µM and 500 µM) decreased cell numbers (P<0.05) and increased both apoptosis (P<0.05) and LC3 protein expression in the blastocysts. Real-time RT-PCR showed that H(2)O(2) significantly decreased mRNA expression of anti-apoptotic gene Bcl-xL but increased pro-apoptotic genes, Caspase 3 (Casp3) and Bak, and autophagy-related genes, microtubule-associated protein 1 light chain 3 (Map1lc3b) and lysosomal-associated membrane protein 2 (Lamp2). However, the addition of H(2)O(2) had no effect on mRNA expression levels in nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1, in blastocysts. These results suggest that H(2)O(2) leads to mitochondrial dysfunction that results in apoptosis and autophagy, which is possibly related to porcine early embryo development.
Department of Animal Sciences, Chungbuk National University, Chungbuk, Korea.
Yong-Nan
YN
Xu
Xiang-Shun
XS
Cui
Shao-Chen
SC
Sun
Seung-Eun
SE
Lee
Ying-Hua
YH
Li
Jung-Suk
JS
Kwon
Sung-Hyun
SH
Lee
Kyu-Chan
KC
Hwang
Nam-Hyung
NH
Kim
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002452:Cell Count; D004477:Ectogenesis / Q000187:drug effects; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D046149:Embryo Culture Techniques; D005786:Gene Expression Regulation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity; D052119:Lysosomal-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D010312:Parthenogenesis; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034421:Sus scrofa
D051017:Apoptosis Regulatory Proteins; D052119:Lysosomal-Associated Membrane Protein 2; D008869:Microtubule-Associated Proteins; D016877:Oxidants; D021122:Protein Subunits; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV