Infection and inflammation of the male reproductive tract are main aetiological factors of infertility. Inflammatory infertility is often based on loss of germ cell function and number. Uropathogenic E. coli (UPEC) is a relevant pathogen in urogenital infections. The aim of this study was to explore the pathomechanism that lead to germ cell loss during E. coli infection. Thus as a first step an in vivo model of epididymo-orchitis was established in rat by injecting a relevant strain of UPEC into the vas deferens in close proximity to the epididymis. Mimicking an infection ascending from the urinary tract, in the testis UPEC bacteria were found exclusively in the interstitial space 7 days post infection. Tracer experiments revealed that the integrity of the blood-testis and blood epididymis barrier was intact 7 days post-infection indicating that evasion of bacteria occurs probably intracellularly. In the testis, UPEC infection resulted in impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+) cells. To investigate potential mechanism of germ cell death, hall mark steps of apoptosis were investigated. Activation of caspase-8 (extrinsic apoptotic pathway), caspase-3/-6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Subsequent investigations focused on in vitro responses of SC and peritubular cells (PTC). Using UPEC wild type and mutant strains, it was shown that only a-hemolysin (+) E. coli strains were able to induce cell death of SC and PTC, but not strains that were lacking this pore forming virulence factor. Subsequent experiments revealed that the caspase-dependent apoptotic pathway was not involved in demise of these cells. Instead nuclear translocation of apoptosis inducing factor (AIF), rapid depletion of ATP, peripheral chromatin aggregation and condensation of nuclei was observed in UPEC treated SC. In contrast, activation of calpain, excessive production of reactive oxygen species (ROS), a gradual decline in ATP levels and the homogenous condensation of nuclei were evident in UPEC induced PTC death. Furthermore, the passive release of high mobility group protein B1 (HMGB1), as an indication of the necrosis, was observed in SC, PTC and infected testis. In summary, the findings indicate that UPEC infection causes the induction of an organized self-destruction cascade termed programmed necrosis in the testis, which may ultimately be the major mechanism contributing to impairment of fertility.
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