Lactate-HIF-1a axis promotes PINK1-dependent mitophagy to protect porcine granulosa cells against hypoxic stress

Ovarian follicular development occurs under chronic hypoxia, imposing significant stress on granulosa cells (GCs) and leading to hypoxia-induced apoptosis, a key factor in follicular atresia and impaired fertility. However, the intrinsic mechanisms enabling GCs to resist hypoxic apoptosis remain unclear. Emerging evidence suggests lactate, beyond its traditional role as a glycolytic byproduct, functions as a critical mediator of cellular stress adaptation. Here, we investigated whether lactate confers protection to porcine granulosa cells (pGCs) by activating mitophagy under hypoxic conditions and elucidated the underlying molecular pathways. Using pGCs subjected to normoxia and hypoxia, combined with pharmacological inhibitors, RNA interference targeting LDHA, HIF-1α, and PINK1, mitochondrial membrane potential assays, apoptosis detection, Western blotting, and mitochondrial-lysosomal co-localization imaging, we demonstrated that lactate depletion exacerbates mitochondrial dysfunction and apoptosis under hypoxia. Conversely, exogenous lactate supplementation attenuated these effects by stabilizing HIF-1α and enhancing PINK1-Parkin-mediated mitophagy. Inhibition of mitophagy via 3-MA or PINK1 knockdown abolished lactate's protective effects, highlighting mitophagy as essential for lactate-mediated cell survival. Cross-species validation in murine granulosa cells (mGCs) in vitro and in vivo confirmed the conservation of lactate's protective mechanisms, where lactate depletion impaired mitochondrial function and increased follicular apoptosis, while lactate supplementation restored mitochondrial integrity and granulosa cell viability. Our findings reveal a novel lactate-HIF-1α-PINK1 signaling axis critical for pGCs resilience to hypoxic stress, offering new therapeutic targets for ovarian disorders such as polycystic ovary syndrome and ovarian aging.