Cycloleucine disturbs functions of porcine Sertoli cell line via modulating gene expression and metabolism

Cycloleucine (CL) is a methyl donor inhibitor. Our previous findings showed that CL could affect meiotic maturation and developmental potency of porcine oocytes by reducing nucleic acid N⁶-methyladenosine (m⁶A) epigenetic modification level. However, the effects of CL on porcine male reproduction are unclear. Here, we showed that CL treatment of porcine testicular Sertoli cell line (SCL) could reduce viability in a dose-dependent manner. CL treatment (40 mM, 36h) of porcine SCL also inhibited proliferation, promoted late apoptosis, increased level of intracellular reactive oxygen species (ROS), reduced mitochondrial function, suppressed levels of nucleic acid N6-methyladenosine (m⁶A), H3K4me3, H3K27ac and H4K16ac, and increased H3K9me2 level. ELISA assays showed that CL (40 mM, 36h) decreased lactate production, but unaltered levels of anti-müllerian hormone. RNA-seq and metabolomics identified 1212 differentially expressed genes (DEGs) (536 up- and 676 down-) and 67 significantly different metabolites (SDMs) (45 up- and 22 down-) to be altered by CL (40 mM, 36h) treatment of porcine SCL. These DEGs and SDMs were involved in multiple pathways, including PI3K-Akt, cell cycle, ferroptosis, FoxO, aminoacyl-tRNA biosynthesis. Validation of some DEGs showed that CL (40 mM, 36h) significantly decreased expression of HMGCS1 and P4HA1, but increased IGFBP5 abundance at both mRNA and protein levels. Combined analysis of transcriptome and metabolome identified the high correlations between some DEGs and SDMs. Collectively, these findings indicate that CL could affect functions of porcine SCL by modifying epigenetic modifications to alter gene expression and metabolism, providing insights into understanding the mechanisms by which epigenetic modification influences the functions of porcine SCL, as well as aiding references for regulating porcine male reproduction.