Involvement of inducible nitric oxide synthase in DNA fragmentation in various testicular germ cells of ethanol-treated rats

Chronic alcohol intake is associated with testicular damage, hypogonadism and infertility. Previously, we reported enhancement of testicular germ cell apoptosis in ethanol-treated rats (ETR) associated with upregulation of apoptosis related genes. Fas, FasL, p53, and activation of caspases. Here, we investigated the involvement of inducible nitric oxide synthase (iNOS) which is a major mediator of oxidative stress in ethanol-induced germ cell apoptosis. Moreover, we explored the various germ cell types undergoing apoptotic DNA fragmentation and their relationship to phagocytosing Sertoli cells. Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. iNOS expression in control and ETR was investigated using immunohistochemistry. DNA fragmentation in various germ cells of ETR was detected using TUNEL method under both light and electron microscopy. Damaged spermatids with apoptotic features were observed by transmission electron microscopy (TEM). Compared to very weak expression of iNOS in control testis, marked increase of iNOS was detected in Sertoli, germ cells and some interstitial cells in ETR. Phagocytosed retained elongated spermatids undergoing DNA fragmentation was detected by TUNEL method in ETR, in addition to spermatids undergoing nuclear vacuolization and chromatin degeneration using TEM. The upregulation of iNOS in the testes of ETR may induce apoptosis of germ cells through the generation of excessive nitric oxide and androgen suppression. Increased DNA fragmentation in spermatids of ETR may be involved in infertility and offspring diseases. We recommend for further research to detect and treat oxidative stress-mediated paternal DNA damage in alcoholics.