Mitochondria-associated endoplasmic reticulum membranes mediate heat stress-induced blood-testis barrier damage via ERS-driven paraptosis in goat Sertoli cells

Abstract Background : Heat stress (HS) severely impairs spermatogenesis and male reproductive performance, primarily by damaging Sertoli cells (SCs) and thereby disrupting the blood-testis barrier (BTB). Paraptosis, a specific form of cell death implicated in stress-induced reproductive disorders, is intimately associated with endoplasmic reticulum stress (ERS), whose activation can be triggered by dysfunction of mitochondria-associated ER membranes (MAMs). However, whether HS induces MAM dysfunction and subsequent ERS-mediated paraptosis in goat SCs remains unclear. Results : In this study, HS exposure impaired spermatozoon quality, induced severe testicular morphological damage, and disrupted BTB integrity in goats, which was accompanied by extensive cytoplasmic single‑membrane vacuolation in SCs. Morphological and molecular evidences collectively verified that this vacuolation was a hallmark of paraptosis in SCs, which was tightly associated with ERS. Meanwhile, HS triggered pronounced ERS and upregulated the PERK signaling pathway in SCs, and inhibition of ERS or PERK significantly attenuated HS-induced SC viability loss and paraptosis. Mechanistically, aberrant expansion of MAMs triggered ERS activation by mediating excessive Ca 2+ transport from the ER to mitochondria via the IP3R-GRP75-VDAC complex, while GRP75 knockdown significantly reversed HS-induced paraptosis by restoring MAM abundance. Further validation confirmed that MAM dysfunction drove HS-induced paraptosis in SCs via modulating the PERK-mediated ERS pathway. Additionally, in vivo validation assays provided consistent evidence that HS exposure resulted in aberrant MAM accumulation, activated PERK-mediated ERS, and induced paraptosis in goat SCs. Conclusion : These results positively confirm that the MAM-ERS-paraptosis regulatory axis governs HS-induced damage to goat SCs, which will facilitate clarifying the underlying mechanism of HS-induced BTB damage and provide potential mitigation strategies for HS-induced male reproductive disorders in goats.