Lipid Droplet Phase Transition in Freezing Cat Embryos and Oocytes Probed by Raman Spectroscopy

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One limitation of cryopreservation is the low tolerance to freezing observed for oocytes and embryos rich in lipid droplets. We apply Raman spectroscopy to investigate freezing of lipid droplets inside cumulus-oocyte complexes, mature oocytes, and early embryos of a domestic cat. Raman spectroscopy allows one to characterize the degree of lipid unsaturation, the lipid phase transition from the liquid-like disordered to solid-like ordered state, and the triglyceride polymorphic state. For all cells examined, the average degree of lipid unsaturation is estimated as ∼1.3 (with ±20% deviation) double bonds per acyl chain. The onset of the lipid phase transition occurs in a temperature range from -10 to +4°C and does not depend on the cell type. Lipid droplets in cumulus-oocyte complexes are found to undergo abrupt lipid crystallization shifted in temperature from the ordering of the lipid conformational state. In the case of mature oocytes and early embryos obtained in vitro, the lipid crystallization is broadened. In the frozen state, lipid droplets inside cumulus-oocyte complexes have a higher content of triglyceride polymorphic β and β' phases than estimated for mature oocytes and early embryos. For the first time, to our knowledge, the temperature evolution of the phase state of lipid droplets is examined. Raman spectroscopy is proved to be a promising tool for in situ monitoring of the lipid phase state in a single embryo/oocyte during its freezing.