Decline in testicular function in ageing rats: Changes in the unfolded protein response and mitochondrial apoptotic pathway

Abstract Activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) and mitochondrial apoptotic pathway serves as a central regulator for maintaining cell function and survival, and is associated with ageing and spermatogenesis. However, changes in UPR activation and mitochondrial apoptotic pathway in the testis during ageing remain unclear. In this study, we hypothesized that UPR activation declines and the mitochondrial apoptotic pathway is activated in the testis during ageing, and these changes are associated with a decline in testicular function. To test this theory, we utilized 6-, 12-, 18-, and 22-month-old Sprague Dawley rats to evaluate the changes in testicular and epididymal weights and indexes, sperm quality, histology, UPR activation, and mitochondrial apoptotic pathway in testicular tissues. The results showed that there was a progressive decline in testicular and epididymal weights and indexes, sperm count, and sperm viability during ageing. Correspondingly, seminiferous tubule diameters and epithelium heights progressively decreased with ageing. Western blot analysis and immunofluorescence staining results revealed that the expression of UPR-related proteins (GRP78, p-PERK, p-eif2α, ATF4, p-IRE1α, ATF6α, and XBP1) progressively decreased in the testis with ageing. In contrast, the expression of ER stress-related pro-apoptotic proteins CHOP, Caspase 12, and p-JNK progressively increased with advancing age. TUNEL staining further confirmed that testicular germ cell apoptosis was significantly increased from month 6 to 22 in rats. Additionally, the relative expression levels of cytochrome c and its downstream molecules including Capsase 9 and Caspase 3 were significantly increased in the testis during ageing. Collectively, our results suggest that impaired UPR activation and increased germ cell apoptosis partly mediated by the UPR and mitochondrial apoptotic pathway might correlate with an age-related decline in testicular function.