Changes in equine testicular and epididymal homeostasis in response to scrotal heating

Heat stress is a common cause of testicular and epididymal dysfunction. While the effect of scrotal heating on ejaculated sperm and testicular histologic changes is known, the testicular and epididymal molecular events and changes in gene expression that cause them are largely unknown. Here, we hypothesized that early equine testicular and epididymal responses to heat stress are associated with oxidative stress and immune dysregulation. Normospermic adult light-breed stallions were divided into two treatments: control (CON, n=5) and scrotal insulation (SI, n=5). The scrotum of SI stallions was covered with an insulation bag for 24 h. The stallions were castrated immediately after the bags were removed. CON stallions did not undergo scrotal insulation. Tissue biopsies were collected from the testes, head, body, and tail of the epididymis for transcriptome analysis. The RNA sequencing libraries were generated using NEBNext^ⓇUltra™RNA Library Prep Kit (Illumina^Ⓡ). Differential gene expression was compared between each SI and CON tissue using the DESeq2 R package and the ClusterProfiler sofware. Differentially expressed genes (DEGs) and pathways were identified (P<0.05, log2 fold change ≥1) in the PANTHER Classification System. The number of DEGs in the testes, head, body, and tail of the epididymis of SI stallions was 507, 1198, 3420, and 1245, respectively. The main downregulated testicular pathways were associated with spermatogenesis. The SI upregulated testicular DEGs corresponded to 331 pathways (268 biological processes, 80.9%, 42 molecular functions, 12.7%, 21 cellular components, 6.3%). Upregulated DEGs were associated with oxidant-antioxidant balance (e.g. peroxidase activity GO:0004601), increased cell respiration, increased use of lipids and amino acids, purine metabolism, activation of the innate and adaptive immune response (e.g. activation of immune response GO:0002253; positive regulation of innate immune response GO:0045089; T cell activation GO:0050863; B cell proliferation GO:0030888; positive regulation of mast cell activation GO:0033005; e.g. FGR, LRRC32, CD226, TLR6, TRIM15, IL33, CD4), and deposition of amyloid. The epididymal head had dysregulation of pathways associated with spermatogenesis, energy metabolism, and antigen presentation and processing, with DEGs associated with macrophage and T cell signaling, regulatory T cell differentiation, and cytotoxic T cells. DEGs in the epididymal body were associated with microtubule formation, sperm flagellum components, and cell motility. DEGs in the epididymal tail were associated with energy metabolism and cell respiration. The findings support dysregulation of the local oxidant-antioxidant system, energy metabolic pathways, and immune system homeostasis in SI stallions and lay the foundation for understanding heat-induced reproductive dysfunction.