Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cyc le and on apoptotic cell death in ovarian cultured granulosa cells. Rates o f cellular proliferation were measured by immunocytochemical staining for p roliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle ana lysis, We also examined for morphological and biochemical signs of apoptosi s. The PCNA expression was downregulated in a dose-dependent manner after t reatment with C-6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C-6-ceramide markedly decreased the populati on associated with G(0)/G(1) DNA content and the reduction of cell numbers in G(0)/G(1) phase was accompanied by the elevation of the A(0) phase. The exposure of granulosa cells to exogenous C-6-ceramide induced drastic morph ological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-a cetate, a protein kinase C (PKC) activator, significantly inhibited the cer amide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G(0)/G(1) phase and as a consequence, gran ulosa cells would be committed to apoptosis. Our findings also indicated th at down-regulation of the PKC activity might be involved in the ceramide-in duced apoptosis in cultured granulosa cells. (C) 1999 Harcourt Publishers L td.