**1. Introduction**

Infertility is a concerning pathophysiological condition that a ffects about 16% of couples worldwide, and approximately half of the cases are attributable to male factors [1]. Even though the cause for the majority of the male infertile cases is unknown, oxidative stress caused by a high amount of reactive oxygen species (ROS) has been observed in 30–80% of infertile patients [2,3]. Oxidative stress is a common feature associated to environmental pollutants, chemotherapeutic agents and other drugs, smoke, toxins, radiation, and diseases such as many types of cancer that have negative e ffects on fertility [4–8]. Even though low ROS levels are required for the acquisition of fertilization ability by the spermatozoon [9], oxidative stress damages spermatozoa by reducing motility, and increasing levels of DNA, protein oxidation and lipid peroxidation [10–12]. As occurring in somatic cells, this oxidative stress observed in spermatozoa is the result of an imbalance between the antioxidant defense system and the endogenous generation of ROS.

Peroxiredoxins (PRDXs) are a family of antioxidant enzymes that are highly expressed from yeas<sup>t</sup> to humans. They are peroxidases that do not require co-factors such as heme group or selenium and contains one or two cysteine (Cys) residues in their active site which are essential for their antioxidant function [13,14]. PRDXs isoforms are divided into 2-Cys PRDXs (PRDX1-4), atypical PRDX (PRDX5) and 1-Cys PRDX (PRDX6). These enzymes are important antioxidants in spermatozoa that regulate the level of ROS such as peroxides (H2O2 and organic hydroperoxides) and peroxinitrite (ONOO−), to avoid cellular toxicity [14,15]. Studies using the PRDX6 knockout mouse model have shown sub-fertility associated with severe impairment of sperm motility and high levels of lipid peroxidation and sperm DNA damage. Interestingly, this abnormal reproductive phenotype worsens with aging [16,17]. Other antioxidant enzymes that fight against oxidative stress in testis and epididymis are catalase, glutathione peroxidases, and thioredoxins [18–20].

Epididymal maturation is a crucial step in the formation of viable and healthy spermatozoa in humans and other mammals [21]. After spermatogenesis, fully formed ye<sup>t</sup> immature and immotile spermatozoa enter into the epididymis, and by the time of exit, they acquire the ability to move and morphological features that optimize their fertilization capacity [21,22]. In addition to sperm maturation, the epididymis also provides essential proteins to spermatozoa via epididymosomes to maintain their cellular functions and to protect them from potential damages such as oxidative stress-dependent injuries [23].

We previously reported that spermatozoa from rats challenged with an in vivo oxidative stress using tert-butyl hydroperoxide (t-BHP), and collected 24 h after the treatment, have higher levels of DNA oxidation and lipid peroxidation and displayed poor motility compared to untreated controls [18]. There was a differential expression of PRDX1 and PRDX6 in a different segmen<sup>t</sup> of the epididymis of the treated rats. Interestingly, these spermatozoa contain high levels of PRDXs, as an attempt of the epididymis to fight against the oxidative stress established by the treatment [18].

In this study, we aimed to elucidate the long-term effect of t-BHP induced oxidative stress on rat reproductive system by assessing the oxidative damage and the expression of significant antioxidants enzymes that fight against hydroperoxides in spermatozoa, epididymis and testis.
