Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs. we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at 39℃, 5% CO2 in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control (6.0±1.0 vs 3.3±0.6, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ERstress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Chemicals    In Vitro Maturation (IVM)    In Vitro fertilization (IVF)    In Vitro Culture (IVC) and Assessment of Embryo Quality    Immunofluorescence Staining    Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-end Labeling    Mitochondrial Staining and Image Analysis    Total RNA Isolation and cDNA Synthesis    Real-time Reverse Transcription-Polymerase Chain Reaction    Statistical Analysis   RESULTS    Effect of 3-MA Treatment on Preimplantation Developmentof Porcine Embryos    Effect of 3-MA Treatment on the LC3-II Expression in Blastocyst Stage Porcine Embryos    Apoptosis and Gene Expression in Blastocyst Stage EmbryosDerived from 3-MA Treated Embryos    Effect of 3-MA on Mitochondrial Distribution in the BlastocystStage Embryos    ER Stress-related Gene (XBP-1) Expression in Cleavage Stage Embryos Derived from 3-MA Treated Embryos   DISUSSION   REFERENCES

• Jin-Mo Park(Department of Biotechnology, College of Engineering, Daegu University)
• Sung-Hun Min(Department of Biotechnology, College of Engineering, Daegu University)
• Joo-Hee Hong(Department of Biotechnology, College of Engineering, Daegu University)
• Enok Lee(Department of Biotechnology, College of Engineering, Daegu University)
• Hyeong-Hoon Son(Department of Biotechnology, College of Engineering, Daegu University)
• Humdai Park(Department of Biotechnology, College of Engineering, Daegu University)
• Deog-Bon Koo(Department of Biotechnology, College of Engineering, Daegu University) Corresponding author