Fas-induced apoptosis in rat thecal/interstitial cells signals through sphingomyelin-ceramide pathway

Of the ovarian follicles that develop during reproductive life, more than 99% do not ovulate and are eliminated from the ovary by follicular atresia. Atresia is achieved by the self destruction of thecal and granulosa cells that comprise the follicle, by the process of apoptosis. The objective of this study was to determine if activation of the Fas receptor could enact apoptosis of thecal cells, and to explore the signal transduction pathway involved. Primary cultures of thecal/interstitial cells isolated from immature rat ovaries were treated with anti-Fas monoclonal antibody (anti-Fas mAb) (2.5 microg/ml). Morphological changes indicative of apoptosis, such as, condensation of chromatin, nucleoplasmic segmentation and formation of apoptotic bodies, were observed by fluorescence microscopy following nucleic acid staining with Hoechst 33342 dye and propidium iodide. DNA analysis of cells after 10 h of treatment with anti-Fas mAb showed that DNA had been cleaved into fragments that were multiples of 180-300 bp in length; biochemical evidence of apoptosis. The sphingomyelin (N-acylsphingosine-1-phosphocholine, SM) pathway that is initiated by the hydrolysis of SM to ceramide (Cer) has been shown previously to be activated by the Fas ligand/receptor system in a number of different cell types. It was therefore possible that the intracellular transduction of Fas receptor activation of thecal/interstitial cells could also involve the SM-Cer pathway. Hence, we have measured the SM levels in control and treated thecal/interstitial cells. Extracts of untreated thecal/interstitial cells contained six major species of SM identified as d18:1/16:0 (sphingosine base/fatty acid), d18:1/18:0, d18:1/20:0, d18:1/22:0, d18:1/24:1, d18:1/24:0 by normal phase high performance liquid chromatography interfaced with electrospray mass spectrometry. Treatment with anti-Fas mAb (2.5 microg/ml) for 30 min caused significant hydrolysis of only two of the SM species, d18:1/16:0 and d18:1/24:1. The involvement of ceramide, the central lipid in this phospholipid second messenger system, was tested using the synthetic cell permeable Cer analog (N-acetyl-N-sphingosine, C2-Cer). C2-Cer (10 microM). This analog induced both morphological and biochemical changes in thecal/interstitial cells, that were characteristic of apoptosis, and the same as those induced by anti-Fas mAb. C2-dihydroceramide (10 microM), an inactive analog of C2-Cer, failed to induce apoptosis of thecal/interstitial cells. In conclusion, the sphingomyelin-ceramide cycle that can lead to cell suicide by apoptosis is functional and activated through the Fas ligand/receptor signal transduction pathway, not only in the immune system, but also in thecal/interstitial cells of the ovarian follicle.