Cross-Talk between Fas/Fas Ligand System and Nitric Oxide in the Pathway Subserving Granulosa Cell Apoptosis: A Possible Regulatory Mechanism for Ovarian Follicle Atresia

• Published in: Endocrinology (Philadelphia) 2005-02, Vol.146 (2), p.808-815
• Main Authors: Chen, Qiumei, Yano, Tetsu, Matsumi, Hirotaka, Osuga, Yutaka, Yano, Naomi, Xu, Jiping, Wada, Osamu, Koga, Kaori, Fujiwara, Toshihiro, Kugu, Koji, Taketani, Yuji
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Biological and medical sciences; Caspase inhibitors; Caspase-3; Caspases - metabolism; Cell activation; Cell culture; Cell Survival - physiology; Cell viability; Cells, Cultured; Cytotoxicity; Fas Ligand Protein; fas Receptor - metabolism; FasL protein; Female; Flow cytometry; Follicles; Follicular Atresia - metabolism; Fundamental and applied biological sciences. Psychology; Granulosa cells; Granulosa Cells - cytology; Granulosa Cells - metabolism; Interferon; Ligands; Membrane Glycoproteins - metabolism; mRNA; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Donors - pharmacology; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type II; Nitric-oxide synthase; Ovaries; Penicillamine - analogs & derivatives; Penicillamine - pharmacology; Rats; Rats, Wistar; Receptor Cross-Talk - physiology; Regulatory mechanisms (biology); Vertebrates: endocrinology; γ-Interferon
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2004-0579

Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-γ, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 μm, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-γ, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.