Background: In mammals, DRP1 is a key regulator of mitochondrial fission during mitochondrial dynamics, whereas ATF5 promotes the mitochondrial unfolded protein response (UPRmt). Both pathways are essential for maintaining cellular homeostasis and protecting oocytes and embryos from external stressors. However, the relationship between ATF5 expression and DRP1 under heat stress conditions during porcine oocyte maturation remains unclear. Methods: In this study, we investigated the mitochondrial dynamics and ATF5 expression in porcine oocytes exposed to heat stress during in vitro maturation (IVM). Protein and gene expression levels were assessed using immunofluorescence staining, Western blotting, and quantitative PCR. Results: During IVM, both DRP1 and ATF5 expression were increased (p < 0.01) significantly. In contrast, heat stress markedly impaired (p < 0.05) meiotic progression and cumulus cell expansion. Mitochondrial dynamics were disrupted (p < 0.05), as fission and fusion markers displayed reciprocal changes relative to those in controls. Concomitantly, the expression of ATF proteins was significantly reduced (p < 0.01) under heat stress. Heat-stressed oocytes also exhibited decreased (p < 0.05) expression of genes involved in antioxidant defense and NAD metabolism, whereas autophagy- and apoptosis-related transcripts were significantly upregulated (p < 0.05). At the blastocyst stage, embryos derived from heat-stressed oocytes exhibited nuclear localization of the UPR-associated transcription factors ATF4, CHOP, and ATF5. Conclusions: Collectively, our findings suggest that heat stress disrupts mitochondrial dynamics and ATF5 expression during porcine oocyte maturation while the UPRmt pathway remains active during early embryonic development to mitigate heat-induced cellular damage.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Chemicals and animals
    Oocyte collection and in vitro maturation (IVM)
    In vitro fertilization and culture (IVF and IVC)
    Orcein staining and assessment of cumulus cellexpansion
    Immunofluorescence (IF) staining
    Western blotting
    Quantitative PCR (qPCR) analysis
    Statistical analysis
RESULTS
    DRP1 and ATF5 expression during IVM of porcineoocytes and heat-stress effects on meioticprogression and cumulus cell expansion
    Changes in the expression of mitochondrial-relatedproteins and genes under heat stress
    Localization of UPRmt-related proteins in blastocystsderived from heat-stressed oocytes
DISCUSSION
CONCLUSION
REFERENCES

• Seul-Gi Yang(Department of Companion Animal Industry, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea)
• Deog-Bon Koo(Department of Companion Animal Industry, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea) Corresponding author
• Hyo-Jin Park(DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea)
• Hun-Wook Ha(DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea)