出版社：American Society for Biochemistry and Molecular Biology
摘要：Ceramide regulates many cellular processes, including cell growth,differentiation, and apoptosis. Although the effects of exogenousbacterial neutral sphingomyelinase (SMase) in Xenopus laevisoocytes have been investigated, its microinjection into oocyteshas not been reported previously. Thus, we compared the incubationversus microinjection of the neutral Bacillus cereus sphingomyelinase(bSMase) to examine whether the topology of ceramide generationdetermines its effects on the fate of oocytes. In agreementwith previous findings, incubation of mature stage VI oocyteswith bSMase increased ceramide levels in oocyte extracts overtime, causing the germinal vesicle breakdown indicative of maturation,without evidence of cytotoxicity. In contrast, bSMase microinjection,which increased ceramide levels in a time- and dose-dependentmanner, resulted in oocyte apoptosis characterized by reactiveoxygen species (ROS) generation, reduced glutathione (GSH) depletionin cytosol and mitochondria, release of cytochrome c and Smac/Diablofrom mitochondria, and caspase-3 activation. Microinjectionof acidic SMase from human placenta recapitulated the apoptoticeffects of bSMase microinjection. Preincubation of oocytes withGSH-ethyl ester before bSMase microinjection prevented ROS generationand mitochondrial downstream events, thus protecting oocytesfrom bSMase-induced death. These findings show a divergent actionof bSMase-induced ceramide on oocyte maturation or apoptosisdepending on the intracellular site where ceramide is generated.Supplementary key words mitochondria • reactive oxygen species • cell death • reduced glutathione
Abbreviations: Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoremethyl coumarin; bSMase, Bacillus cereus sphingomyelinase; GCS, glucosylceramide synthase; GSH, reduced glutathione; GVBD, germinal vesicle breakdown; hSMase, human placenta sphingomyelinase; NSMase, neutral sphingomyelinase; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; ROS, reactive oxygen species; SMase, sphingomyelinase; TNF, tumor necrosis factor
