Acute Heat Stress Remodels Mitochondrial Ultrastructure and Time-Dependent Transcriptomic Landscapes in Bovine Granulosa Cells

Heat stress is a critical environmental factor that compromises reproductive performance in dairy cattle, primarily through deleterious effects on ovarian follicular cells. Granulosa cells (GCs), which provide essential nutritional and hormonal support for oocyte maturation, are particularly vulnerable to hyperthermic insult. However, the early molecular and subcellular events that precede overt cellular dysfunction remain inadequately characterized. This study aimed to investigate the temporal dynamics of mitochondrial morphological alterations and transcriptomic reprogramming in bovine GCs subjected to acute in vitro heat stress. Bovine GCs were exposed to 43 °C for 10, 20, 30, or 40 min, with untreated cells serving as controls. Heat shock protein expression (HSP90 and HSPA1A) was evaluated by qRT-PCR. Intracellular calcium concentration was measured using the Fluo-3 AM fluorescent probe. Mitochondrial ultrastructure was examined via transmission electron microscopy (TEM). Global transcriptional changes were profiled by RNA sequencing (RNA-seq) at 20 and 40 min post-treatment, followed by bioinformatics analysis. Nine selected differentially expressed genes (DEGs) were validated by qRT-PCR. HSP90 and HSPA1A expression was significantly upregulated at 30 and 40 min of heat exposure (p < 0.05). Intracellular Ca2+ concentration exhibited a biphasic pattern—markedly elevated at 20 min but significantly reduced at 40 min. TEM revealed progressive mitochondrial swelling and cristae disruption at both time points, with more severe damage at 40 min. RNA-seq identified 185 DEGs at 20 min and 831 DEGs at 40 min, with 285 genes commonly dysregulated across both time points. Functional enrichment analysis demonstrated that the 20-min response was dominated by heat shock protein-related pathways, whereas the 40-min response expanded to encompass apoptosis, inflammation, steroidogenesis, and ciliogenesis pathways. qRT-PCR validation confirmed the reliability of the RNA-seq data. These findings provide a mechanistic framework for understanding heat stress-induced ovarian dysfunction and may inform strategies to preserve fertility in dairy cattle during thermal challenge.