ABSTRACT
BACKGROUND: We wanted to identify the presence of the estrogen receptor (ER) alpha in Sertoli cells and gain insight on the regulation of the ER alpha gene expression by testosterone in Sertoli cells. The transcriptional regulation of the ER alpha gene was investigated in primary Sertoli cell cultures by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Primary Sertoli cell culture was performed. The expression levels of ER alpha and ER beta mRNA in Sertoli cells were detected by Northern blot, RT-PCR, immunocytochemistry and in situ hybridization. RESULTS: The ovary, testis and epididymis showed a moderate to high expression of ER alpha while the prostate, ovary and LNCap cells showed the ER beta expression. ER alpha mRNA and protein were detected in the germ cells and Sertoli cells by in situ hybridization and immunocytochemistry. The level of ER alpha mRNA was gradually decreased in a time-dependent manner after testosterone treatment, and the changes of ER alpha mRNA were dependent on the concentration of testosterone. Androgen binding protein and testosterone-repressive prostate message-2 (TRPM-2) mRNA were reduced at 24 hour by estradiol, while the transferrin mRNA was not affected. ER alpha mRNA was strongly detectable in the testes of 7 days-old-rats, but it was gradually decreased from 14 to 21 days of age. The primary Sertoli cells also showed the same pattern. The ER alpha gene expression was also regulated by testosterone in the Sertoli cells prepared from the 14- and 21-day old rats. CONCLUSIONS: These results suggest that ER alpha is transcriptionally regulated by testosterone and it may play some role in the Sertoli cells.
Subject(s)
Animals , Female , Male , Rats , Androgen-Binding Protein , Blotting, Northern , Cell Culture Techniques , Epididymis , Estradiol , Estrogen Receptor alpha , Estrogens , Gene Expression , Germ Cells , Immunohistochemistry , In Situ Hybridization , Ovary , Prostate , RNA, Messenger , Sertoli Cells , Testis , Testosterone , TransferrinABSTRACT
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.
Subject(s)
Animals , Rats , fas Receptor , 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, Sprague-Dawley , Response Elements , RNA, Messenger , Testis , Testosterone , Weights and MeasuresABSTRACT
BACKGROUNDS: Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated. METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay. RESULTS: The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor(P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration. CONCLUSION: These results suggest that the Fas/FasL system and Bcl-2 may be important to castrationdependent apoptosis in the rat ventral prostate, with SP-1 related to the castration-dependent regulation of the FasL gene
Subject(s)
Animals , Rats , fas Receptor , Apoptosis , Binding Sites , Blotting, Western , Castration , Cell Death , Deoxyribonuclease I , DNA , DNA Footprinting , DNA Fragmentation , Electrophoretic Mobility Shift Assay , Epithelial Cells , Fas Ligand Protein , In Situ Nick-End Labeling , Prostate , Rats, Sprague-Dawley , RNA, Messenger , Testosterone , Tolonium ChlorideABSTRACT
BACKGROUNDS: Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated. METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay. RESULTS: The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor(P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration. CONCLUSION: These results suggest that the Fas/FasL system and Bcl-2 may be important to castrationdependent apoptosis in the rat ventral prostate, with SP-1 related to the castration-dependent regulation of the FasL gene