Effects of ceramide, the Fas signal intermediate, on apoptosis and phospholipase D activity in mouse ovarian granulosa cells in vitro

Although recent studies have demonstrated that ovarian follicular atresia o ccurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized, We examin ed the role of ceramide as a candidate intracelluar mediator of Fas mediate d signaling in cultured granulosa cells. Expression of Fas antigen was demo nstrated by Western blot of granulosa cell lysates and immunostaining of cu ltured granulosa cells, Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, w hich was accompanied by a progressive increase in endogenous levels of cera mide, The addition of exogenous C-6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradati on. Furthermore, both anti-Fas mAb and C-6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a d ose-dependent manner, In addition, treatment with phorbol 12-myristate 13-a cetate completely attenuated the ceramide-induced inhibition of PLD activit y and partially suppressed ceramide-induced apoptosis, These results indica te that the Fas/ceramide signaling pathway might play a role in granulosa c ell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.