Peroxiredoxin 4 Involved in Spermatogenesis by Affecting Oxidative Stress and Ferroptosis

The main functions of testes are sperm production and androgen secretion in testicular befitting microenvironment. Excessive level of the reactive oxygen species (ROS) from metabolism and cellular events can lead to oxidative stress (OS) and ferroptosis, which injure the functions of mitochondria and endoplasmic reticulum (ER) of testicular cells, leading to the impaired spermatogenesis and spermiogenesis, as well as insufficient androgen. Our previous studies have shown that peroxiredoxin 4 (Prdx4) is a vital ER-located protector against endoplasmic reticulum stress (ERS) in ovarian granulosa cells. In this study, we explored whether Prdx4 played a beneficial role in testes, and the potential application value in male reproduction.

Prdx4 knockout mice and Prdx4^-/y Leydig cells were established using CRISPR/Cas9 system, the natural adult (9 weeks) mice, aging (9 months) mice, and wild-type MLTC-1 cells were used as control. To further investigate the protective effect of Prdx4 in the adult mice, the testicular 43°C water-bath was used to induce an OS model.

The male Prdx4^-/y mice aged over 9 months showed the age-related pathology of seminiferous tubule, the increased OS and ERS, and the decreased fertility. The adult Prdx4^-/y mice under heat stress manifested increased levels of OS, including almost 1.5-fold rise in 4-hydroxynonenal (4-HNE) and 8-hydroxy-2'- deoxyguanosine (8-OHdG), higher ferroptosis, and poorer fertility. Prdx4^-/y Leydig cells showed a higher level of ferroptosis after hydrogen peroxide treatment. ACSL4 increased by 18.6% while COX2 increased by 13.5%, and the expressions of GPX4 and SLC7A11 decreased by 49.2% and 24.3%, respectively. Increased levels of ferroptosis in MLTC-1 leads to mitochondria-derived OS and apoptosis, which can be partially restored by deferoxamine and overexpressing SLC7A11.

We concluded that Prdx4 as a protective factor plays a critical role in preserving spermatogenesis by alleviating testicular OS and ferroptosis, and that Prdx4 may be known as a potential target in mitigating the adverse effects of natural aging and OS injury.