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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

Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, Tokyo 113-8655, Japan

Address all correspondence and requests for reprints to: Dr. Tetsu Yano, Associate Professor, Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: tetu-tky{at}umin.ac.jp.

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-, 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-, 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.

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