Acid ceramidase is a novel factor required for early embryo survival

Abstract

Recent studies suggest that the lipid, ceramide, induces the default apoptosis process in eggs. Yet, it is obscure how newly formed embryos overcome this fate. Acid ceramidase (AC) is a key regulatory enzyme involved in ceramide metabolism, and mutations in the AC gene (Asah1) result in Farber Lipogranulomatosis, a fatal human genetic disorder. Our previous studies revealed that AC knockout (Asah1-/-) mice had a lethal phenotype, and herein we reveal the mechanism underlying this observation. A single-cell, polymerase chain reaction (PCR) genotyping method was developed to analyze individual embryos from Asah1 +/- intercrosses. Combined with Annexin V staining, this genotype analysis demonstrated that Asah1-/- embryos could not survive beyond the 2-cell stage, and underwent apoptotic death. Notably, sphingosine-1-phosphate (S1P) treatment of early 2-cell embryos from the Asah1 +/- intercrosses rescued Asah1-/- embryos, and enabled their progression from the 2-cell to 4-8-cell stage. Quantitative PCR also revealed that expression of the Asah1 gene in healthy embryos was initiated at the 2-cell stage, coincident with embryonic genome activation (EGA). AC activity and Western blot analyses further demonstrated high expression and activity of the enzyme in normal, unfertilized eggs, which likely provide the protein to newly formed embryos prior to EGA. Based on these observations, we suggest that AC is an essential factor required for embryo survival that functions by removing ceramide from the newly formed embryos, thus inhibiting the default apoptosis pathway.