Integrated Multi-Omics Reveals Disruption of Lipid–Steroid Regulatory Axis Underlying Chronic Heat Stress-Induced Ovarian Dysfunction in Mice

Heat stress is an escalating environmental challenge that adversely affects mammalian reproductive physiology, yet the integrated molecular mechanisms through which ovarian tissue responds to sustained heat stress remain incompletely understood. This study investigated the effects of chronic heat stress on mouse ovarian tissue using an integrated multi-omics approach. Female C57BL/6 mice were exposed to heat stress at 42 °C for 20 min daily over 30 days. Whole ovarian tissues were subsequently analyzed using RNA sequencing, DIA-based proteomics, untargeted metabolomics, histological examination, and RT-qPCR validation. Histological analysis showed a significant reduction in secondary follicle profiles in heat-stressed mice, whereas primary and antral follicle profiles showed inter-animal variability and did not differ significantly between groups. Transcriptomic and proteomic analyses identified altered expression of lipid metabolism- and steroidogenesis-related molecules, including Apoa1, Apoa2, Lpl, Fads2, and Hsd17b2. Metabolomic profiling further showed marked changes in metabolites related to lipid metabolism and steroid hormone biosynthesis. Integrated pathway analysis revealed recurrent enrichment of pathways associated with lipid metabolism and endocrine regulation, particularly PPAR signaling, cholesterol metabolism, and steroid hormone biosynthesis. RT-qPCR validation confirmed altered expression of selected genes involved in lipid transport and steroidogenic pathways. These findings suggest that chronic heat stress may disrupt ovarian metabolic and endocrine homeostasis through alterations in lipid–steroid regulatory pathways. This study provides systems-level evidence supporting the involvement of interconnected metabolic pathways in heat stress-associated ovarian dysfunction.