Autophagy Inhibitor, 3-Methyladenine, Reduces Preimplantation Development and Blastocyst Qualities in Pigs

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 35권 / 3호
ㆍ저자명 : Jin-Mo Park, Sung-Hun Min, Joo-Hee Hong, Enok Lee, Hyeong-Hoon Son, Humdai Park, Deog-Bon Koo

목차

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

영어 초록

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.

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