TNFα /TNFα receptors in adult rat testis after ethylene dimethane sulfonate treatment (609.15)

Abstract

Tumor necrosis factor (TNF‐α) is a multifunctional cytokine secreted in mammalian testis by germ cells, Sertoli cells and to some extent by macrophage; however TNF‐α receptors are largely confined to Sertoli cells. TNF‐α regulates different cellular processes pertinent to spermatogenesis including steroidogenesis, germ cell apoptosis, growth hormone function and inflammation. The Fas/FasL apoptotic pathway is the current model for depletion of Leydig cells with ethylene dimethane sulfonate (EDS) treated adult rat testis. We are investigating a potential role of TNF‐α in EDS Leydig cell depletion as well as other tissue correlates. At 6, 15 and 24 hr post‐EDS, using RT‐ and q‐PCR we find that mRNA expression of Leydig cell markers, Lhr and Insl‐3 declined by more than 85% and 3β‐HSD declined by 97% at 24 hr after EDS compared to controls. Using TUNEL, a DNA fragmentation detection assay, we observed a time dependent loss of Leydig cells by apoptosis. Next, genes associated with inflammation, TNF‐α was increased modestly and IL‐1β increased 3 fold, 24 hr post‐EDS. A three‐fold increase in TNF‐α receptor superfamily member 1A mRNA (Tnfrsf1a/ CD120a) was found. However, no change was observed in tumor necrosis factor receptor superfamily member 1B mRNA (Tnfrsf1b/CD120b) at 24 hr post‐ treatment. The mRNA of the two TNF‐α receptors has a differential expression, suggesting their presence on different cell types. ELISA detection of the testicular tissue showed an increased TNF‐α protein expression at 24 hr post‐EDS. Thus, with a high percentage of Leydig cell loss at a time when TNF‐α and its receptors are changing may imply TNF‐α activity is involved with Leydig cell apoptosis following EDS, however, stronger correlates may be found with other cell types. Future work is to localize TNF‐α receptors using immunohistochemistry.