2024년 한국동물생명공학회 학술 대회 [Dong-Woo Kim] > 학회발표

Zearalenone induces severe ER-stress through the IRE1 derived ER-phagy in preimplantation porcine embryos in vitro

Dong-Woo Kim^1,2, Hyo-Jin Park^1,2, Seul-Gi Yang^1,2, Eun-Seo Kim^1,2, Hun-Wook Ha^1,2, Deog-Bon Koo^1,2,3

¹ Department of Biotechnology, College of Engineering, ² Center for Infertility, ³ Department of Companion Animal Industry, College of Natural and Life Sciences, Daegu University, Gyeongsan 38453, Korea

Zearalenone (ZEN) a mycotoxin commonly found in animal feed is known to disrupt porcine embryo development during in vitro culture(IVC). However, the mechanism of the inositol-requiring enzyme 1 (IRE1) derived ER-phagy response to the endoplasmic reticulum (ER) stress by ZEN exposure on porcine embryo developmental capacity during IVC remains unclear. First, porcine embryos were incubated in IVC medium supplemented with 2.5, 5, and 10 μM ZEN for 2 days to confirm the blastocyst development competence. As a result, exposure to ZEN led to a significant dose-dependent reduction (p < 0.05) in blastocyst development rates, the formation of expanded blastocysts, and the total number of nuclei in porcine embryos. Simultaneously, ZEN treated embryos resulted in a significant increase (p < 0.001) in DNA fragmentation and fluorescence intensity of mitochondrial and intracellular Ca2+ levels in blastocysts, as confirmed by TUNEL assay, Rhod-2 AM staining, and Fluo-4 AM staining. ER-tracker was used to measure ER distribution  in  porcine  blastocysts  treated  with  5  and  10 μM  ZEN.  ZEN  exposure  decreased  (p < 0.001) ER fluorescence intensity and increased the number of blastocysts with uneven cytoplasmic distribution. We examined whether ZEN treatment induces ER stress-related autophagy in porcine embryos by analyzing the co-expression of LC3B, FAM134B, and ER-tracker. Western blot results confirmed significant increases in protein levels of IRE1 pathway components (GRP78, p-IRE1, and IRE1) and the autophagy marker LC3B in ZEN-treated porcine embryos (p < 0.05), indicating activation of the unfolded protein response (UPR) signaling pathway. In addition, the protein level of ER-phagy  receptor  protein  (FAM134B)  was  significantly  decreased in porcine blastocysts treated with ZEN. In conclusion, ZEN exposure in porcine inhibits IRE1-mediated ER-phagy, leading to a significant increase in ER stress and apoptosis, ultimately impairing embryonic developmental capacity.

*This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2021R1C1C2009469, NRF-2022R1A2C1002800, and NRF-2021R1A6A3A01087623) funded by and the Ministry of Science and ICT, Republic of Korea.

Key words: ZEN, ER-stress, IRE1 signaling, ER-phagy, Pig