Myclobutanil-induced testis damage and apoptotic germ cell death through ER stress and autophagy in mouse testes

Myclobutanil (MYC) is an effective triazole fungicide used to control fungal diseases in crops, but concerns have been raised about its reproductive toxicity at high concentrations. In this study, we evaluated the effects of MYC on the male reproductive system using in vivo and in vitro models. Oral administration of 150 mg/kg MYC to male mice for 8 weeks (five times a week) significantly reduced testis size and the populations of major testicular cell types, including differentiated germ cells, Sertoli cells, and Leydig cells. Gene expression in germ cells (DDX4, SYCP3, DMC1, STRA8), Sertoli cells (SOX9, WT1, AMH), and Leydig cells (STAR, CYP17A1, 3β-HSD1, 17β-HSD3, INSL3) was suppressed by MYC. Sperm motility indices (motility, VSL, LIN, STR) were significantly decreased. Immunohistochemical analysis showed that cells positive for GRP78/BiP (ER stress), LC3A/B (autophagy), and Cleaved Caspase-3 (apoptosis) were mainly distributed along the luminal border of the seminiferous tubules in MYC-treated testes. MYC was examined in GC1-spermatogonia (spg) to find markers associated with ER stress, autophagy, and apoptosis. MYC treatment induced dose-dependent cytotoxicity and increased the expression of ER stress markers (GRP78/BiP, PDI, IRE1), autophagy markers (ATG5, BECN1, ULK1), and apoptosis markers (BAX, BAD, Cleaved Caspase-3) in GC1-spg treated with 100 µM MYC. Inhibition of autophagy using 3-methyladenine (3-MA) attenuated MYC-induced apoptosis, confirming a mechanistic link between autophagy and GC1-spg. These results demonstrate that MYC induces testicular toxicity through ER stress and autophagy-mediated germ cell apoptosis, highlighting the potential reproductive risks associated with MYC exposure.