Ethanol Exposure Enhances Apoptosis Within the Testes

Background: Chronic ethanol abuse causes testicular atrophy and male infertility in alcoholic men. It is well known that ethanol exposure disrupts the hypothalamic‐pituitary‐gonadal axis, adversely affects the secretory function of Sertoli cells, and produces oxidative stress within the testes. It is still not clear what cellular mechanisms are responsible for the morphologic alteration of the testes that results in a reduction of testicular mass as a consequence of ethanol exposure. The hypothesis tested was that ethanol enhances apoptosis of testicular germ cells. Methods: In the experiments of chronic ethanol exposure, male Sprague Dawley® rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) were fed Liber‐Decarlie liquid diet for 9 weeks. In the experiments of acute ethanol exposure, a small volume of 20% ethanol solution was administered by intratesticular injection. Both 3′‐end labeling of isolated testicular deoxyribonucleic acid (DNA) and labeling of apoptotic cells in situ by the terminal deoxynucleotidyl transferase‐mediated deoxyuridine 5′‐triphosphate nick end‐labeling method were used to determine apoptosis rates within the testes. The expression of proteins involved in apoptosis was assessed by reverse transcription‐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 spermatogonia as well as spermatocytes. Direct intratesticular injections of ethanol solution enhanced testicular DNA fragmentation, suggesting an increase in apoptosis. Moreover, Fas ligand levels were increased within the testes of rats that were chronically fed ethanol. In vitro, ethanol treatment of cultured Sertoli cells enhanced the production of Fas ligand. In addition, testicular levels of p53 messenger ribonucleic acid were increased in rats that were chronically fed ethanol. Conclusions: All of these observations suggest that ethanol enhances testicular germ cell apoptosis.