OBJECTIVE: One of the models we study is the ovarian granulosa cell (GC), a somatic cell lineage that is critically important for maintenance of the female germ line and many endocrine functions of the ovaries. The objective of this study is to clarify the significance of ceramide and the role of ceramide metabolism in dictating the fate of cells exposed to stress. METHODS: We first treated GC with a C8-ceramide analog or an amine derivative of ceramide that cannot be metabolized by ceramidase (C8-ceramine). Northern blot analysis was performed to evaluate mRNA of acid ceramidease expression regualted by gonadotropin and in situ hybridization was done to identify the mRNA expression of acid ceramidase in ovaries. RESULTS: After 6 hours, C8-ceramide (50 micro M) triggered apoptosis in only 28 +/- 6% of the cells, whereas C8-ceramine (50 micro M) induced apoptosis in all cells (LD50=1 micro M). These data suggested that ceramidase activity is a critical determinant of GC survival. In situ hybridization showed that mRNA of acid ceramidase was highly expressed in GC in growing follicle. mRNA of acid ceramidase was expressed abundantly in granulosa cells and ovaries and its expression was significantly increased by gonadotropin in granulosa cells in in situ hybridization. Forty two hour after gonadotropin treatment, mRNA expression of acid ceramidase in granulosa cells was two fold increased cells comparing with no treatment control in northern blot analysis (P<0.05). In copora lutea, elevated mRNA expression of acid ceramidase was decreased. CONCLUSION: We concluded that GC possess inherently high levels of ceramidase activity, and that ceramidase has important for metabolizing ceramind to maintain GC survival in the ovary.
Acid Ceramidase ; Apoptosis ; Blotting, Northern ; Cell Lineage ; Ceramidases* ; Female ; Germ Cells ; Gonadotropins ; Granulosa Cells* ; Humans ; In Situ Hybridization ; Metabolism ; Ovary ; RNA, Messenger