IRE1/FAM134B-mediated ER-phagy alleviates zearalenone-induced ER stress and developmental defects in porcine embryos

Zearalenone (ZEN), a mycotoxin commonly found in animal feed, impairs the female reproductive function owing to its estrogen-mimicking ability. ZEN exposure induces endoplasmic reticulum (ER) stress, oxidative stress, and autophagy activation. However, the involvement of inositol-requiring enzyme 1 (IRE1)-mediated ER-phagy in ZEN-induced ER stress and embryonic competence during in vitro culture (IVC) remains unclear. Porcine embryos were cultured with 2.5, 5, and 10 μM ZEN for two days after in vitro fertilization (IVF) to assess blastocyst development. ZEN exposure caused a significant dose-dependent decrease in blastocyst formation, expanded blastocysts, and total cell number (p < 0.05). ZEN-treated embryos exhibited increased DNA fragmentation along with elevated mitochondrial and intracellular Ca²⁺ levels (p < 0.001), as confirmed using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and Rhod-2 and Fluo-4 AM staining. ER-tracker analysis revealed decreased ER fluorescence intensity and increased uneven cytoplasmic ER distribution in blastocysts exposed to 5 and 10 μM ZEN (p < 0.001). Western blotting showed activation of the unfolded protein response (UPR) with increased expression of glucose-regulated protein 78 (GRP78), p-IRE1, IRE1, p-c-Jun N-terminal kinase (JNK), JNK, C/EBP homologous protein (CHOP), and the autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B) (p < 0.05), whereas the ER-phagy receptor family with sequence similarity 134, member B was downregulated. Tauroursodeoxycholic acid (TUDCA) alleviated ZEN-induced ER stress, restoring ER distribution and calcium homeostasis, reducing DNA damage, and improving blastocyst development. These findings suggest that ZEN disrupts IRE1 signaling and suppresses ER-phagy during early porcine embryo development, whereas TUDCA alleviates ER stress and improves embryonic competence during IVC.