Korean J Urol.  2005 Jul;46(7):750-760.

Mechanism of Azaline B-induced Apoptosis in Rat Testis

• Park C
• Yun EJ
• Song KS
• Kim YR
• Kim JS
• Lee SD
• Park JI
• Lee JH
• Park SK
• Hwang BD
• Lim K

Abstract

PURPOSE: To identify the mechanism of azaline B-dependent apoptosis, the regulation of Fas and FasL genes has been investigated.
MATERIALS AND METHODS
Azaline B was subcutaneously injected into Sprague-Dawley rats. The levels of Fas receptor (Fas) and Fas ligand (FasL) were detected by reverse transcription-polymerase chain reaction (RT- PCR). Azaline B-dependent apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) and DNA fragmentation assay. Transacting factor of FasL promoter was identified by DNase I footprinting and DNA mobility shift assay.
RESULTS
The azaline B-treated testis (250microgram/kg body wt/day) had decreased to 70+/-2.5% and 38+/-1.8% of the normal testis weight at 3 and 5 days after the injection, respectively, but the weights of the testis were not changed after pretreatment of follicle-stimulating hormone (FSH) and testosterone. Apoptosis of the testis was detected by DNA fragmentation assay and TUNEL assay after the azaline B treatment. The levels of Fas and FasL mRNA were increased by the treatment of azaline B in both time- and dose-dependent manners. In DNase I footprinting assay with FasL promoter, the nuclear factor prepared from control was bound with at least four sites: SP-1 binding site at 283, NF-kappa B binding site at 219, TATA at 132 and the gamma-interferon response element (gamma-IRE) at 78. gamma-IRE was completely protected by the nuclear extract prepared from azaline B-treated rat testis. In DNA mobility shift assay, the binding activity of gamma-IRE binding protein was increased after azaline B treatment.
CONCLUSIONS
These results suggest that Fas-FasL system may be important to azaline B-dependent apoptosis in rat testis and that gamma-IRE binding protein is related to the azaline B-dependent regulation of FasL gene.

Keyword

Azaline; Fas receptor; Fas ligand (FasL); Apoptosis

MeSH Terms

Animals
Antigens, CD95
Apoptosis*
Binding Sites
Carrier Proteins
Deoxyribonuclease I
DNA
DNA Fragmentation
Electrophoretic Mobility Shift Assay
Fas Ligand Protein
Follicle Stimulating Hormone
In Situ Nick-End Labeling
Interferon-gamma
NF-kappa B
Rats*
Rats, Sprague-Dawley
Response Elements
RNA, Messenger
Testis*
Testosterone
Weights and Measures
Antigens, CD95
Carrier Proteins
DNA
Deoxyribonuclease I
Fas Ligand Protein
Follicle Stimulating Hormone
Interferon-gamma
NF-kappa B
RNA, Messenger
Testosterone