Fig 2: Morphological changes and ER stress responses after testicular hyperthermia in mice. (A) WT and DKO (Line 80) mice were exposed to testicular hyperthermia (HS). After 1 or 4 weeks, the sperm counts of the mice in the indicated genotype were determined (n = 3 for each group). Data are the mean ± SEM. Statistical analyses were performed using two-way ANOVA with Tukey's test. Different letters indicate statistically significant differences (p < 0.05). (B) WT and DKO (Line 80) mice were exposed to testicular hyperthermia (HS). After 1 or 4 weeks, the testis/body weight ratio of the mice of the indicated genotype was determined (n = 3 for each group). Data are the mean ± SEM. Statistical analyses were performed using two-way ANOVA with Tukey's test. Different letters indicate statistically significant differences (p < 0.05). (C) WT and DKO (Line 80) mice were exposed to testicular hyperthermia (HS). After 1 or 4 weeks, PAS staining for testicular morphology for the indicated genotype are shown. The sections were counterstained with Mayer's hematoxylin. Scale bar, 200 µm. The right panel depicts the quantification of mean tubule area (n = 4 for each group). Data are the mean ± SEM. Statistical analyses were performed using two-way ANOVA with Tukey's test. Different letters indicate statistically significant differences (p < 0.05). (D) WT and DKO (Line 80) mice were exposed to testicular hyperthermia (HS). After 1 week, testes were collected and the protein production levels of ATF4, CHOP, PRDX4, PRDX4t, SPA17, GPX4, and ß-actin in testes were determined by western blotting (n = 3 for each group). The graph depicts the quantification of ATF4, CHOP, and GPX4 normalized to the corresponding ß-actin. Data are the mean ± SEM. Statistical analyses were performed using two-way ANOVA with Tukey's test. Different letters indicate statistically significant differences (p < 0.05).

Fig 3: Schematic explanation of cisplatin-induced testicular injury and the counteracting protective effects of nanosome honokiol. Normal testis (in grey box) consists of sertoli cells forming tight junction (black bar) to maintain testicular structure. Multi-layered spermatogenic cells represent normal and ongoing spermatogenesis with different stages of maturating sperm cells. Cisplatin (red box) induces excessive intracellular ROS production that subsequently lead to Bip-ATF4-CHOP ER stress. Additionally, excessive ROS causes DNA breaks in the testis and results in caspase 3-associated cellular apoptosis. Uncontrolled ROS production activates TGFß1 and upregulates TXNDC5 which subsequently increase aSMA and type I collagen protein expression for fibroblast activation and extracellular matrix accumulation in the testis. All- above mentioned damages result in testis dysfunction and compromise normal sperm production. Nanosome honokiol (in green box) acts as ROS scavenger with its known anti-oxidation bio-property, reduction of cisplatin-induced ROS not only significantly attenuates Bip-ATF4-CHOP ER stress and testicular DNA breaks, but also reduce caspase 3-associated cell death. By down regulation of TGFß1 and ER protein TXNDC5, fibroblast activation and type I collagen accumulation were both mitigated. Improvement of testicular environment attributes to the restoration of sperm production and normal testicular functions.