The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

ABSTRACT   
INTRODUCTION   
MATERIALS AND METHODS    
ocyte Collection and In Vitro Maturation    Embryo Culture    
Terminal Deoxynucleotidyl Transferase-Mediated dUTPNick-End Labeling (TUNEL) 
AssaTerminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) 
Assayy    
Mitochondrial Staining and Image Analysis    
Immunofluorescence Staining    
Real Time Reverse Transcription Polymerase Chain Reaction(Real Time RT-PCR)    
Statistical Analysis   
RESULTS    
Effect of Different Concentrations of Glucose on PorcineEmbryos Developing In Vitro    
Glucose Concentration Affects Cell Number and Apoptosisin The Blastocyst Stage    
Hyperglycemia Affects Mitochondrial Distribution but not Nuclear DNA-Encoded Mitochondrial Gene Expressionin Blastocysts    
Effect of Hyperglycemia on Autophagy and Autophagy-Related Gene Expression in Blastocysts   
DISCUSSION   
REFERENCES

• Yong-Nan Xu(Department of Animal Sciences, Chungbuk National University)
• Ying-Hua Li(Department of Animal Sciences, Chungbuk National University)
• Sung Hyun Lee(Department of Animal Sciences, Chungbuk National University)
• Jung-Woo Kwon(Department of Animal Sciences, Chungbuk National University)
• Seul Ki Lee(Department of Animal Sciences, Chungbuk National University)
• Young-Tae Heo(Department of Animal Sciences, Chungbuk National University)
• Xiang-Shun Cui(Department of Animal Sciences, Chungbuk National University)
• Nam-Hyung Kim(Department of Animal Sciences, Chungbuk National University) Corresponding author