Alcohol Induced Apoptosis on Leydig Cell Line, TM3

Kim, Khae Hawn; Kwon, Chil Hun; Park, Heung Jae

Abstract

PURPOSE
It is well known that alcoholics with hepatic cirrhosis exhibit alterations in the hypothalamic-pituitary-gonadal axis as well as vitamins and trace element deficiencies leading to an inadequate testicular function, which is related to impotence and infertility. Several studies on chronic alcoholics have revealed that alcohol exerts cytotoxic effect at the testicular level. However, the exact mechanisms of alcohol cytotoxicity on testicular cells have not been clarified. In this study, it was determined whether or not alcohol induces apoptosis in Leydig cells.
MATERIALS AND METHODS
The Leydig cells, TM3, were grown in culture. The cells were exposed to various alcohol (Ethanol) concentrations for 3 hours, ranging from 50mM to 500mM. The cells without alcohol treatment served as the controls. The cell viability and proliferative activities were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The apoptotic cellular changes were assessed by 4,6-diamidino-2-phenylindole (DAPI) staining, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, which were confirmed with a DNA fragmentation assay. A further investigation with reverse transcription-polymerase chain reaction (RT-PCR), caspase-3 enzyme assay, and Western blot analysis was performed.
RESULTS
The MTT assay revealed that alcohol induces Leydig cell cytotoxicity in a concentration dependent manner. Alcohol treatment resulted in definitive apoptotic features including DNA fragmentation, increased caspase-3 enzyme activity, mRNA expression, and protein.
CONCLUSIONS
In this study, alcohol was shown to induces typical apoptosis on Leydig cells via caspase-3 activation. It is suggested that alcohol-induced apoptosis is one of the important mechanisms in male urogenital disorders including erectile dysfunction and infertility. Further studies to clarify the detailed mechanisms of alcohol-induced apoptosis on Leydig cells are recommended.