Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes

Oocyte cryopreservation has great potentials in the reproductive technologies but it is an open challenge in the majority of animals and little is known on the biochemical transformation induced by this process in the cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. Our aim was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein structure from the α-helices to the β-sheet form, while lipids tend to assume a packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. The modifications were not observed in oocytes exposed to CPs, suggesting that they do not affect the biochemical architecture of the oocyte. In oocytes exposed to CPs decreased developmental competence and increased ROS production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes suggest a degree of reversibility of the induced transformations, which renders vitrified oocytes similar to untreated cells after 2 h warming.