Ethanol exposure enhances apoptosis within the testes

Background: Chronic ethanol abuse causes testicular atrophy and male infert ility in alcoholic men. It is well known that ethanol exposure disrupts the hypothalamic-pituitary-gonadal axis, adversely affects the secretory funct ion of Sertoli cells, and produces oxidative stress within the testes. It i s still not clear what cellular mechanisms are responsible for the morpholo gic alteration of the testes that results in a reduction of testicular mass as a consequence of ethanol exposure. The hypothesis tested was that ethan ol enhances apoptosis of testicular germ cells. Methods: In the experiments of chronic ethanol exposure, male Sprague Dawle y(R) rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) were fed Liber-De carlie liquid diet for 9 weeks. In the experiments of acute ethanol exposur e, a small volume of 20% ethanol solution was administered by intratesticul ar injection. Both 3'-end labeling of isolated testicular deoxyribonucleic acid (DNA) and labeling of apoptotic cells in situ by the terminal deoxynuc leotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labelin g method were used to determine apoptosis rates within the testes. The expr ession of proteins involved in apoptosis was assessed by reverse transcript ion-polymerase chain reaction and by Western blotting. Results: The testes of rats that were fed an ethanol-containing liquid diet had more testicular DNA fragmentation than did those of animals that were fed an isocaloric control diet. Ethanol increased the number of apoptotic s permatogonia as well as spermatocytes. Direct intratesticular injections of ethanol solution enhanced testicular DNA fragmentation, suggesting an incr ease in apoptosis. Moreover, Fas Ligand levels were increased within the te stes of rats that were chronically fed ethanol. In vitro, ethanol treatment of cultured Sertoli cells enhanced the production of Fas ligand. In additi on, testicular levels of p53 messenger ribonucleic acid were increased in r ats that were chronically fed ethanol. Conclusions: All of these observations suggest that ethanol enhances testic ular germ cell apoptosis.