Elevated peritoneal fluid ceramides in human endometriosis-associated infertility and their effects on mouse oocyte maturation.

OBJECTIVE: To characterize the peritoneal fluid (PF) sphingolipid profile in endometriosis-associated infertility (EAI), and to assess the plausible functional role(s) of ceramides in oocyte maturation potential. DESIGN: Retrospective case-control study and in vitro mouse oocyte study. SETTING: University-affiliated hospital and university laboratory. SUBJECTS: Twenty-seven infertile patients diagnosed with endometriosis and 20 infertile patients who did not have endometriosis; BALB/c female mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): PF sphingolipid concentrations. Number of metaphase II (MII) mouse oocytes. RESULT(S): Liquid chromatography-tandem mass spectrometry revealed 11 significantly elevated PF sphingolipids in infertile women with severe endometriosis compared with infertile women without endometriosis (change >50%, false discovery rate ≤10%). Logistic regression analysis identified three very-long-chain ceramides potentially associated with EAI. Functional studies revealed that very-long-chain ceramides may compromise or induce murine MII oocyte maturation. The oocyte maturation effects induced by the very long-chain ceramides were triggered by alterations in mitochondrial superoxide production in a concentration-dependent manner. Scavenging of mitochondrial superoxide reversed the maturation effects of C24:0 ceramide. CONCLUSION(S): EAI is associated with accumulation of PF very-long-chain ceramides. Mouse studies demonstrated how ceramides affect MII oocyte maturation, mediating through mitochondrial superoxide. These results provide an opportunity for direct functional readout of pathophysiology in EAI, and future therapies targeted at this sphingolipid metabolism may be harnessed for improved oocyte maturation.

Long-chain glucosylceramides crosstalk with LYN mediates endometrial cell migration.

Endometriosis is a disease characterized by regurgitated lesions which are invasive and migratory, embedding at ectopic, extra-uterine locations. Extracellular glucosylceramides (GlcCers), bioactive sphingolipids potentiating signals for cell migration, are found in elevated levels in endometriosis; however underlying mechanisms that result in cellular migration are poorly defined. Here, we demonstrated that internalized GlcCer induced migratory activity in immortalized human endometrial stromal cells (HESCs), with highest potency observed in long-chain GlcCer. Long-chain ceramide (Cer) similarly induced cellular migration and mass spectrometry results revealed that the migratory behavior was contributed through glycosylation of ceramides. Cells treated with GlcCer synthase inhibitor, or RNAi-mediated knockdown of glucosylceramide synthase (GCS), the enzyme catalyzing GlcCer production attenuated cell motility. Mechanistic studies showed that GlcCer acts through stromal cell-derived factor-1 alpha and its receptor, CXC chemokine receptor 4 (SDF-1α-CXCR4) signaling axis and is dependent on phosphorylation of LYN kinase at Tyr396, and dephosphorylation of Tyr507. Migration was prominently attenuated in cells exposed to CXCR4 antagonist, AMD3100, yet can be rescued with diprotin A, which prevents the degradation of SDF-1α. Furthermore, blocking of LYN kinase activity in the presence of SDF-1α and GlcCer reduced HESC migration, suggesting that LYN acts downstream of GlcCer-SDF-1α-CXCR4 axis as part of its intracellular signal transduction. Our results reveal a novel role of long-chain GlcCer and the dialog between GlcCer, LYNpTyr396 and SDF-1α-CXCR4 in inducing HESC migration. This finding may improve our understanding how endometriotic lesions invade to their ectopic sites, and the possibility of using GlcCer to modulate the SDF-1α-CXCR4-LYNpTyr396 axis in endometriosis.