Effects of Zearalenone on Autophagy in the Testes of Male Mice at Different Developmental Stages

Zearalenone (ZEA) is an estrogenic mycotoxin that poses a potential threat to male reproductive health. This study investigated the age-dependent testicular toxicity of ZEA and its association with autophagy-related changes in male mice. Male C57BL/6J mice at different developmental stages, TM3 cells, and primary ovine Leydig cells were used to evaluate the effects of ZEA on testicular morphology, testosterone secretion, autophagy-related protein expression, and signaling pathways. ZEA induced seminiferous tubule disorganization and spermatogenic cell sloughing in 4–8-week-old mice, whereas no obvious testicular pathological changes were observed in 2-week-old mice. Serum testosterone levels showed a downward trend following ZEA exposure, although the differences were not statistically significant. Western blotting revealed changes in the expression of autophagy-related proteins in testicular tissue, including decreased levels of Beclin1 and LC3, accompanied by increased phosphorylation of PI3K and AKT at 2, 6, and 8 weeks of age and significantly increased mTOR phosphorylation at 6 and 8 weeks of age. In vitro, 40 μM ZEA significantly reduced the viability of TM3 and primary ovine Leydig cells and altered the expression of autophagy-related proteins. These changes were accompanied by increased phosphorylation of PI3K and AKT, whereas the increase in mTOR phosphorylation was not statistically significant in either cell model. Collectively, these findings indicate that susceptibility to ZEA-associated testicular toxicity varies with developmental stage, with more evident histopathological changes observed in 4–8-week-old mice. ZEA exposure was also associated with alterations in autophagy-related protein expression and PI3K/AKT/mTOR pathway phosphorylation. Further studies are needed to determine whether ZEA alters autophagic flux and whether these signaling changes causally contribute to testicular toxicity and Leydig cell dysfunction.