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Sphingolipid metabolism in Xenopus laevis oocyte maturation and apoptotic death
Author(s) -
Petcoff Douglas W.,
Bulow Aviva A.,
Hostetter Trevor D.,
Stith Bradley J.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb222
Subject(s) - xenopus , phosphorylation , microbiology and biotechnology , ceramide , biology , oocyte , apoptosis , programmed cell death , sphingolipid , cycloheximide , biochemistry , embryo , protein biosynthesis , gene
Physiological maturation of Xenopus laevis oocytes is brought about by progesterone (PG). The action of PG involves an increase in ceramide and can be mimicked by injection of ceramide or treatment with sphingomyelinase (SMase)(Strum et al. 1995). Whereas prophase‐arrested Xenopus oocytes can be maintained in culture for several days, matured oocytes or eggs (both MII‐arrested) die rather quickly. The apoptotic death of maturing Xenopus oocytes has recently been shown to correlate with increased phosphorylation of Bad on residue S128 (Du Pasquier et al. 2011). We have initiated a study to determine the relationship between sphingolipid metabolism, Bad S128 phosphorylation, and cell death in Xenopus oocytes. We have found that treating cells with SMase triggers maturation and death in a manner that is similar to the effects of PG treatment. These events correlate to an increase in phosphorylation of Bad S128 as evidenced by western blotting with an anti‐S128 Bad phosphospecific antibody. Phosphorylation of this residue is slowed by including the JNK inhibitor SP600125 in the assay. To assess the post‐mitochondrial phase of apoptosis, we have used a microinjected near‐infrared dye caspase substrate to show that treatment with ceramide increases the activity of caspase 3. In characterizing the roles of SM pathways, JNK activity, and Bad S128 phosphorylation state in the biochemistry of maturing oocytes, we are currently using both genetic and pharmacologic approaches to teasing apart maturation and death. For example, using cycloheximide we have shown that both PG and SMase‐induced maturation require protein synthesis, whereas phosphorylation of Bad S128 does not. Results of experiments currently under way using genetic manipulation of JNK and sphingosine kinase 1 (SK1) will also be reported.