Lipid droplet phase transition in freezing cat embryos and oocytes probed by Raman spectroscopy

Abstract

ABSTRACTEmbryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One challenging limitation of this technology is the cell damage during freezing associated with the intracellular lipid droplets. We exploit a Raman spectroscopy to investigate the freezing of cumulus-oocyte complexes, mature oocytes and early embryos of a domestic cat. All these cells are rich in lipids. The degree of lipid unsaturation, lipid phase transition from liquid-like disordered to solid-like ordered state (main transition) and triglyceride polymorphic state are studied. For all cells examined, the average degree of lipid unsaturation is estimated about 1.3 (with ±20 % deviation) double bonds per acyl chain. The onset of the main lipid phase transition occurs in a temperature range from −10 to +4 °C and does not depend significantly on the cell type. It is found that lipid droplets in cumulus-oocyte complexes undergo an abrupt lipid crystallization, which not completely correlate with the ordering of lipid molecule acyl chains. In the case of mature oocytes and early embryos obtainedin vitrofrom cumulus-oocyte complexes, the lipid phase transition is broadened. In frozen state lipid droplets inside the cumulus-oocyte complexes have higher content of triglyceride polymorphic β and β′ phases (∼66%) than it is estimated for the mature oocytes and the early embryos (∼50%). For the first time, to our knowledge, temperature evolution of lipid droplets phase state is examined. Raman spectroscopy is proved as a prospective tool forin situmonitoring of lipid phase state in single embryo/oocyte during freezing.