Heat stress induces mitochondrial dysfunction and ATF5-mediated UPR ^mt activation via GRP75-dependent ER-mitochondria coupling in bovine embryos

Background: Heat stress (HS), driven by global climate change, adversely affects reproductive performance in livestock and mammals by impairing gamete development and reducing fertility.Exposure of oocytes to elevated temperatures during in vitro maturation (IVM) can induce persistent cellular stress that compromises subsequent embryonic development.However, the involvement of ATF5-mediated UPR mt signaling and GRP75-associated ER-mitochondria communication during early bovine embryogenesis under HS remains unclear.Methods: In this study, bovine cumulus-oocyte complexes (COCs) were matured for 22 h at either 38.5℃ (control; Con) or 41.5℃ (HS), followed by in vitro fertilization and embryo culture under standard conditions.Results: HS exposure during IVM significantly reduced cleavage and blastocyst formation rates (p < 0.01).Embryos derived from HS-exposed oocytes exhibited decreased mitochondrial membrane potential (ΔΨm), increased reactive oxygen species (ROS) production, and mitochondrial calcium (Ca 2+ ) accumulation, suggesting disruption of GRP75 (known as HSPA9)-mediated ER-mitochondria communication.These embryos also showed increased expression of HSPA9 and HSPD1 at the cleavage stage, indicating persistence of stress signals beyond fertilization.Immunofluorescence analysis revealed enhanced nuclear localization of ATF5 in blastocysts, while qPCR analysis demonstrated increased expressions of ATF5-mediated UPR mt -related genes, including LONP1, HSPD1, and HSPA9.Conclusions: Collectively, these findings demonstrate that heat stress during oocyte maturation induces persistent mitochondrial dysfunction associated with GRP75mediated mitochondrial Ca 2+ dysregulation and ATF5-mediated UPR mt activation, which may represent an adaptive compensatory response in surviving bovine embryos.